Long-acting polypeptides and methods of producing and administering same

ABSTRACT

A polypeptide and polynucleotides comprising at least two carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a non-human peptide-of-interest are disclosed. Pharmaceutical compositions comprising the non-human polypeptides and polynucleotides of the invention and methods of using both human and non-human polypeptides and polynucleotides are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/309,496, filed Jun. 19, 2014, which is (1) a continuation in part ofU.S. patent application Ser. No. 14/059,134, filed Oct. 21, 2013, whichis a continuation in part of U.S. patent application Ser. No.13/804,354, filed Mar. 14, 2013 and issued Feb. 3, 2015 as U.S. Pat. No.8,946,155, which is a continuation in part of U.S. patent applicationSer. No. 13/192,542 filed Jul. 28, 2011 and issued Jun. 18, 2013 as U.S.Pat. No. 8,465,948, which is a continuation of U.S. patent applicationSer. No. 12/401,755 filed Mar. 11, 2009 and issued Feb. 14, 2013 as U.S.Pat. No. 8,114,836, which is a continuation of U.S. application Ser. No.11/702,156 filed on Feb. 05, 2007, now abandoned, which claims thebenefit of U.S. Provisional Application Ser. No. 60/764,761, filed Feb.3, 2006, and (2) a continuation-in-part of U.S. patent application Ser.No. 13/195,931, filed Aug. 2, 2011 and issued May 28, 2013 as U.S. Pat.No. 8,450,269, which is a continuation-in-part of U.S. patentapplication Ser. No. 12/509,188, filed Jul. 24, 2009 and issued Nov. 6,2012 as U.S. Pat. No. 8,304,386, which is a continuation-in-part of U.S.patent application Ser. No. 12/476,916, filed Jun. 2, 2009 and issuedNov. 1, 2011 as U.S. Pat. No. 8,048,849, which is a continuation-in-partof U.S. patent application Ser. No. 12/401,746, filed Mar. 11, 2009 andissued Jan. 17, 2012 as U.S. Pat. No. 8,097,435, and is a continuationof U.S. patent application Ser. No. 11/700,910, filed Feb. 1, 2007 andissued Jun. 30, 2009 as U.S. Pat. No. 7,553,940, which claims thebenefit of U.S. Provisional Application Ser. No. 60/764,761, filed Feb.3, 2006. All of these applications are hereby incorporated by referencein their entirety herein.

FIELD OF INVENTION

A polypeptide and polynucleotides encoding same comprising at least twocarboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to apeptide-of-interest are disclosed. Pharmaceutical compositionscomprising the polypeptide and polynucleotides of the invention andmethods of using same are also disclosed.

BACKGROUND OF THE INVENTION

Polypeptides are susceptible to denaturation or enzymatic degradation inthe blood, liver or kidney. Accordingly, polypeptides typically haveshort circulatory half-lives of several hours. Because of their lowstability, peptide drugs are usually delivered in a sustained frequencyso as to maintain an effective plasma concentration of the activepeptide. Moreover, since peptide drugs are usually administered byinfusion, frequent injection of peptide drugs causes considerablediscomfort to a subject. Thus, there is a need for technologies thatwill prolong the half-lives of therapeutic polypeptides whilemaintaining a high pharmacological efficacy thereof. Such desiredpeptide drugs should also meet the requirements of enhanced serumstability, high activity and a low probability of inducing an undesiredimmune response when injected into a subject.

Unfavorable pharmacokinetics, such as a short serum half-life, canprevent the pharmaceutical development of many otherwise promising drugcandidates. Serum half-life is an empirical characteristic of amolecule, and must be determined experimentally for each new potentialdrug. For example, with lower molecular weight polypeptide drugs,physiological clearance mechanisms such as renal filtration can make themaintenance of therapeutic levels of a drug unfeasible because of costor frequency of the required dosing regimen. Conversely, a long serumhalf-life is undesirable where a drug or its metabolites have toxic sideeffects.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method of reducing thedosing frequency of a growth hormone in a subject, comprisingadministering to said subject a therapeutically effective amount of apolypeptide consisting of a growth hormone, one chorionic gonadotrophincarboxy terminal peptide (CTP) attached to the amino terminus of saidgrowth hormone, and two chorionic gonadotrophin CTPs attached to thecarboxy terminus of said growth hormone, and wherein said polypeptideoptionally consists of a signal peptide attached to the amino terminusof said one CTP, thereby reducing the dosing frequency of a growthhormone in a subject.

In another embodiment, the invention relates to a method of improvingthe area under the curve (AUC) of a growth hormone in a subject,comprising administering to said subject a therapeutically effectiveamount of a polypeptide consisting of a growth hormone, one chorionicgonadotrophin carboxy terminal peptide (CTP) attached to the aminoterminus of said growth hormone, and two chorionic gonadotrophin CTPsattached to the carboxy terminus of said growth hormone, and whereinsaid polypeptide optionally consists of a signal peptide attached to theamino terminus of said one CTP, thereby reducing the dosing frequency ofa growth hormone in a subject.

In one embodiment, the invention relates to a method of treating asubject in need of GH therapy, comprising administering to said subjecta therapeutically effective amount of a polypeptide consisting of agrowth hormone, one chorionic gonadotrophin carboxy terminal peptide(CTP) attached to the amino terminus of said growth hormone, and twochorionic gonadotrophin CTPs attached to the carboxy terminus of saidgrowth hormone, and wherein said polypeptide optionally consists of asignal peptide attached to the amino terminus of said one CTP, therebyreducing the dosing frequency of a growth hormone in a subject.

In another embodiment, the invention relates to a method of increasinginsulin-like growth factor (IGF-1) levels in a subject, comprisingadministering to said subject a therapeutically effective amount of apolypeptide consisting of a growth hormone, one chorionic gonadotrophincarboxy terminal peptide (CTP) attached to the amino terminus of saidgrowth hormone, and two chorionic gonadotrophin CTPs attached to thecarboxy terminus of said growth hormone, and wherein said polypeptideoptionally consists of a signal peptide attached to the amino terminusof said one CTP, thereby increasing insulin-like growth factor (IGF-1)levels in a subject.

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are diagrams illustrating six EPO-CTP constructs.

FIG. 1A—is a diagram of the polypeptide of SEQ ID NO: 1.

FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2.

FIG. 1C is a diagram of the polypeptide of SEQ ID NO: 3.

FIG. 1D is a diagram of the polypeptide of SEQ ID NO: 4.

FIG. 1E is a diagram of the polypeptide of SEQ ID NO: 5.

FIG. 1F is a diagram of the polypeptide of SEQ ID NO: 6.

FIG. 2 is a photograph illustrating the expression of the EPO-CTPvariants from transfected DG44 cells. Final test samples fromtransfected cells were prepared as described under “sample preparation”and run on SDS/PAGE. Proteins were detected by Western blot.

FIG. 3 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-3 (SEQ ID NO: 3). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-3,rhEPO-WT (SEQ ID NO: 16), Recormon® (Commercial EPO) or Recormon® (5μg/kg) 3 times a week. Control animals were injected i.v. with PBS.Blood samples were collected three times a week and haematocrit levelswere detected. Each point represents the group average of haematocrit(%) ±SE.

FIG. 4 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-1 (SEQ ID NO: 1). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-1,rhEPO-WT (SEQ ID NO: 16), Recormon® or Recormon® (5 μg/kg) 3 times aweek. Control animals were injected i.v. with PBS. Blood samples werecollected three times a week and haematocrit levels were detected. Eachpoint represents the group average of haematocrit (%) ±SE.

FIG. 5 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-2 (SEQ ID NO: 2). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-2 (SEQ IDNO: 2), rhEPO-WT (SEQ ID NO: 16), Recormon® or Recormon® (5 μg/kg) 3times a week. Control animals were injected i.v. with PBS. Blood sampleswere collected three times a week and haematocrit levels were detected.Each point represents the group average of haematocrit (%) ±SE.

FIG. 6 is a time graph illustrating the change in reticulocyte levelfollowing a single bolus dose of EP0-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp®.

FIG. 7 is a time graph illustrating the change in hemoglobin level(presented as change from baseline) following a single bolus dose ofEP0-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp®.

FIG. 8 is a time graph illustrating the change in hematocrit levelfollowing a single bolus dose of EP0-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp®.

FIG. 9 is a graph illustrating the change in serum concentration ofEPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp® post i.v.injection.

FIG. 10 is a Western blot illustrating the molecular weight & identityof MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO: 37), MOD-4022 (SEQ IDNO: 38), MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40). A pAGESDS gel was blotted and stained using monoclonal anti-hGH antibodies.The photograph indicates that like commercial and wild type hGH,MOD-7020-4 variants are recognized by anti-hGH antibodies.

FIG. 11 is a bar graph illustrating the weight gain of hypophysectomizedrats following administration of the GH-CTP polypeptides of the presentinvention.

FIG. 12 are graphs showing the mean plasma CTP-hGH-CTP-CTP or GHconcentrations (pg/ml) following a single i.v. or s.c. dose ofCTP-hGH-CTP-CTP or GH in rats (n=3-6 per dose/route).

FIG. 13 are graphs showing the mean incremental weight gain following asingle s.c. doses of CTP-hGH-CTP-CTP (0.4, 0.8 and 4 mg/Kg) inhypophysectomized rats in comparison to daily GH injections (0.1mg/Kg/Day) (n=10 per dose).

FIG. 14 is a graph showing the area Under the Curve following singleinjection of CTP-hGH-CTP-CTP correlates with Body Weight gain in Rats.

FIG. 15 is a graph showing the incremental weight gain following an s.c.doses of CTP-hGH-CTP-CTP (0.4, 0.8 and 4 mg/Kg) 4 days apart inhypophysectomized rats in comparison to daily GH injections (0.1mg/Kg/Day) (n=10 per dose).

FIG. 16 is a graph showing hGH serum concentration in hypophysectomizedrat following SC injection of CTP-hGH-CTP-CTP and commercial hGH. Singledose of CTP-hGH-CTP-CTP 0.6 or 1.8 mg/Kg and Biotropin 0.35 or 1.05mg/Kg were injected subcutaneously to hypophysectomised rats fordetermination of PK/PD profile. Serum hGH post injection was measuredusing specific ELISA kits.

FIG. 17 is a graph showing IGF-1 serum levels in Hypophysectimized RatsFollowing SC injection of CTP-hGH-CTP-CTP and commercial hGH. Singledose of CTP-hGH-CTP-CTP 0.6 or 1.8 mg/Kg and Biotropin 0.35 or 1.05mg/Kg were injected subcutaneously to hypophysectomised rats fordetermination of PK/PD profile. Serum IGF-I post injection was measuredusing specific ELISA kits (Roche Diagnostics).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention describes long-actingpolypeptides and methods of producing and using same. In anotherembodiment, long-acting polypeptides comprise carboxy terminal peptide(CTP) of human Chorionic Gonadotropin (hCG). In another embodiment, CTPacts as a protectant against degradation of proteins or peptides derivedtherefrom. In another embodiment, CTP extends circulatory half-lives ofproteins or peptides derived therefrom. In some embodiments, CTPenhances the potency of proteins or peptides derived therefrom.

In another embodiment, “CTP peptide,” “carboxy terminal peptide,” and“CTP sequence” are used interchangeably herein. In another embodiment,the carboxy terminal peptide is a full-length CTP. In anotherembodiment, the carboxy terminal peptide is a truncated CTP. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, “signal sequence” and “signal peptide” are usedinterchangeably herein. In another embodiment, “sequence” when inreference to a polynucleotide can refer to a coding portion. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, “peptide of interest” and “polypeptide sequenceof interest” are used interchangeably herein. In another embodiment, thepeptide of interest is a full-length protein. In another embodiment, thepeptide of interest is a protein fragment. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the invention provides a polypeptide consisting agrowth hormone, a single chorionic gonadotrophin carboxy terminalpeptide attached to the amino terminus of the growth hormone, and twochorionic gonadotrophin carboxy terminal peptides attached to thecarboxy terminus of the growth hormone. In another embodiment, theinvention provides a polypeptide consisting a growth hormone, a singlechorionic gonadotrophin carboxy terminal peptide attached to the aminoterminus of the growth hormone, two chorionic gonadotrophin carboxyterminal peptides attached to the carboxy terminus of the growthhormone, and a signal peptide attached to the amino terminus of onechorionic gonadotrophin carboxy terminal peptide.

In another embodiment, a growth hormone comprising CTPs as describedherein has enhanced in vivo biological activity compared the same growthhormone without CTPs. In another embodiment, a growth hormone comprisingat least one CTP attached to its amino terminus and at least two CTPsattached to its carboxy terminus has enhanced in vivo biologicalactivity compared the same growth hormone without CTPs. In anotherembodiment, a growth hormone comprising one CTP attached to its aminoterminus and two CTPs attached to its carboxy terminus has enhanced invivo biological activity compared the same growth hormone without CTPs.

In another embodiment, a polypeptide comprising at least twocarboxy-terminal peptide (CTP) sequences of chorionic gonadotrophinattached to a polypeptide sequence of interest, wherein a first CTPsequence of the at least two CTP sequences is attached to an aminoterminus of the polypeptide sequence of interest and a second CTPsequence of the at least two CTP sequences is attached to the carboxyterminus of the polypeptide sequence of interest is provided. In anotherembodiment, the carboxy-terminal peptide (CTP) sequence is of humanchorionic gonadotrophin.

In another embodiment, a subject is a human subject. In anotherembodiment, a subject is a pet. In another embodiment, a subject is amammal. In another embodiment, a subject is a farm animal. In anotherembodiment, a subject is a dog. In another embodiment, a subject is acat. In another embodiment, a subject is a monkey. In anotherembodiment, a subject is a horse. In another embodiment, a subject is acow. In another embodiment, a subject is a mouse. In another embodiment,a subject is a rat. In one embodiment, the subject is male. In anotherembodiment, the subject is female.

The phrase “polypeptide sequence of interest” refers, in anotherembodiment, to any polypeptide sequence, such as one comprising abiological activity. In another embodiment, the peptide is glycosylated.In another embodiment, the peptide is non-glycosylated. Examples ofpolypeptides which benefit from an extension in their circulatoryhalf-life include, but are not limited to erythropoietin (EPO),interferons, human growth hormone (hGH) and glucagon-like peptide-1(GLP-1).

In another embodiment, the configuration of CTP-growth hormone-CTP-CTPas described herein comprises a growth hormone or an active fragmentthereof connected via a peptide bond to at least one CTP unit. Inanother embodiment, a CTP-growth hormone-CTP-CTP as described hereincomprises a growth hormone or an active fragment thereof connected via apeptide bond to at least one CTP unit which is connected to anadditional CTP unit via a peptide bond. In another embodiment, apolypeptide as described herein comprising a growth hormone fragmentsthereof and CTP units and/or fragments thereof are interconnected via apeptide bond. In another embodiment, one nucleic acid molecule encodes apolypeptide as described herein comprising a growth hormone and/orfragments thereof and CTP units and/or fragments thereof.

In another embodiment, the carboxy-terminal peptide (CTP) is attached tothe polypeptide sequence of interest via a linker. In anotherembodiment, at least one CTP is optionally attached to said polypeptidesequence of interest via a linker. In another embodiment, the linkerwhich connects the CTP sequence to the polypeptide sequence of interestis a covalent bond. In another embodiment, the linker which connects theCTP sequence to the polypeptide sequence of interest is a peptide bond.In another embodiment, the linker which connects the CTP sequence to thepolypeptide sequence of interest is a substituted peptide bond. Inanother embodiment, the carboxy-terminal peptide (CTP) sequencecomprises an amino acid sequence selected from the sequences set forthin SEQ ID NO: 48.

In another embodiment, SEQ ID NO: 48 comprises the following amino acid(AA) sequence: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILQ (SEQ ID NO: 48).

In another embodiment, the carboxy terminal peptide (CTP) of humanChorionic Gonadotropin (hCG) is fused to a protein. In anotherembodiment, the carboxy terminal peptide (CTP) of human hCG is fused toa glycoprotein. In another embodiment, the carboxy terminal peptide(CTP) of hCG is fused to a glycoprotein hormone. In another embodiment,the CTP of hCG is fused to a peptide derived from a glycoproteinhormone. In some embodiments, glycoprotein hormones comprise EPO, FSH,or TSH and peptides derived therefrom.

In some embodiments, a CTP sequence at both the amino terminal end of apolypeptide and at the carboxy terminal end of the polypeptide provideenhanced protection against degradation of a protein. In someembodiments, CTP sequences at both the amino terminal end of apolypeptide and at the carboxy terminal end of the polypeptide providean extended half-life to the attached protein.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of thepolypeptide, and at least one additional CTP sequence attached in tandemto the CTP sequence at the carboxy terminus provide enhanced protectionagainst degradation of a protein. In some embodiments, a CTP sequence atthe amino terminal end of a polypeptide, a CTP sequence at the carboxyterminal end of the polypeptide, and at least one additional CTPsequence attached in tandem to the CTP sequence at the carboxy terminusprovide an extended half-life to the attached protein. In someembodiments, a CTP sequence at the amino terminal end of a polypeptide,a CTP sequence at the carboxy terminal end of the polypeptide, and atleast one additional CTP sequence attached in tandem to the CTP sequenceat the carboxy terminus provide enhanced activity of the attachedprotein.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of thepolypeptide, and at least one additional CTP sequence attached in tandemto the CTP sequence at the amino terminus provide enhanced protectionagainst degradation of the attached protein. In some embodiments, a CTPsequence at the amino terminal end of a polypeptide, a CTP sequence atthe carboxy terminal end of the polypeptide, and at least one additionalCTP sequence attached in tandem to the CTP sequence at the aminoterminus provide an extended half-life to the attached protein. In someembodiments, a CTP sequence at the amino terminal end of a polypeptide,a CTP sequence at the carboxy terminal end of the polypeptide, and atleast one additional CTP sequence attached in tandem to the CTP sequenceat the amino terminus provide enhanced activity the attached protein.

In some embodiments, a CTP sequences at both the amino terminal end of agrowth hormone and at the carboxy terminal end of the growth hormoneprovide enhanced protection against degradation of a growth hormone. Inanother embodiment, at least one CTP sequence at the amino terminal endof a growth hormone and two CTP units in the carboxy terminal end of agrowth hormone provide enhanced protection against clearance. In anotherembodiment, at least one CTP sequence at the amino terminal end of agrowth hormone and two CTP units in the carboxy terminal end of a growthhormone provide prolonged clearance time. In another embodiment, atleast one CTP sequence at the amino terminal end of a growth hormone andtwo CTP units in the carboxy terminal end of a growth hormone enhanceC_(max) of a growth hormone. In another embodiment, at least one CTPsequence at the amino terminal end of a growth hormone and two CTP unitsin the carboxy terminal end of a growth hormone enhance T_(max) of agrowth hormone. In another embodiment, at least one CTP sequence at theamino terminal end of a growth hormone and two CTP units in the carboxyterminal end of a growth hormone enhanced T1/2.

In some embodiments, CTP sequences at both the amino terminal end of agrowth hormone and at the carboxy terminal end of the growth hormoneextend the half-life of the modified growth hormone. In anotherembodiment, at least a single CTP sequence at the amino terminal end ofa growth hormone and at least two CTP sequences at the carboxy terminalend of the growth hormone provide an extended half-life to the modifiedgrowth hormone. In another embodiment, a single CTP sequence at theamino terminal end of a growth hormone and two CTP sequences at thecarboxy terminal end of the growth hormone provide extended half-life tothe attached growth hormone. In another embodiment, a single CTPsequence at the amino terminal end of a growth hormone and two CTPsequences in tandem at the carboxy terminal end of the growth hormoneprovide extended half-life to the modified growth hormone.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of the growthhormone, and at least one additional CTP sequence attached in tandem tothe CTP sequence at the carboxy terminus provide enhanced protectionagainst degradation to a growth hormone. In some embodiments, a CTPsequence at the amino terminal end of a growth hormone, a CTP sequenceat the carboxy terminal end of the growth hormone, and at least oneadditional CTP sequence attached in tandem to the CTP sequence at thecarboxy terminus extend the half-life of the growth hormone. In someembodiments, a CTP sequence at the amino terminal end of a growthhormone, a CTP sequence at the carboxy terminal end of the growthhormone, and at least one additional CTP sequence attached in tandem tothe CTP sequence at the carboxy terminus enhance the biological activityof the growth hormone.

In another embodiment, the carboxy terminal peptide (CTP) peptide of thepresent invention comprises the amino acid sequence from amino acid 112to position 145 of human chorionic gonadotrophin, as set forth in SEQ IDNO: 17. In another embodiment, the CTP sequence of the present inventioncomprises the amino acid sequence from amino acid 118 to position 145 ofhuman chorionic gonadotropin, as set forth in SEQ ID NO: 18. In anotherembodiment, the CTP sequence also commences from any position betweenpositions 112-118 and terminates at position 145 of human chorionicgonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29,30, 31, 32, 33 or 34 amino acids long and commences at position 112,113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

In another embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 1-5 conservativeamino acid substitutions as described in U.S. Pat. No. 5,712,122. Inanother embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 1 conservativeamino acid substitution. In another embodiment, the CTP peptide is avariant of chorionic gonadotrophin CTP which differs from the native CTPby 2 conservative amino acid substitutions. In another embodiment, theCTP peptide is a variant of chorionic gonadotrophin CTP which differsfrom the native CTP by 3 conservative amino acid substitutions. Inanother embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 4 conservativeamino acid substitutions. In another embodiment, the CTP peptide is avariant of chorionic gonadotrophin CTP which differs from the native CTPby 5 conservative amino acid substitutions. In another embodiment, theCTP peptide amino acid sequence of the present invention is at least 70%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 80% homologous to the native CTP aminoacid sequence or a peptide thereof. In another embodiment, the CTPpeptide amino acid sequence of the present invention is at least 90%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 95% homologous to the native CTP aminoacid sequence or a peptide thereof.

In another embodiment, the CTP peptide DNA sequence of the presentinvention is at least 70% homologous to the native CTP DNA sequence or apeptide thereof. In another embodiment, the CTP peptide DNA sequence ofthe present invention is at least 80% homologous to the native CTP DNAsequence or a peptide thereof. In another embodiment, the CTP peptideDNA sequence of the present invention is at least 90% homologous to thenative CTP DNA sequence or a peptide thereof. In another embodiment, theCTP peptide DNA sequence of the present invention is at least 95%homologous to the native CTP DNA sequence or a peptide thereof.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is truncated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are truncated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are truncated. In one embodiment, the truncated CTP comprisesthe first 10 amino acids of SEQ ID NO: 43. In one embodiment, thetruncated CTP comprises the first 11 amino acids of SEQ ID NO: 43. Inone embodiment, the truncated CTP comprises the first 12 amino acids ofSEQ ID NO: 43. In one embodiment, the truncated CTP comprises the first13 amino acids of SEQ ID NO: 43. In one embodiment, the truncated CTPcomprises the first 14 amino acids of SEQ ID NO: 43. In one embodiment,the truncated CTP comprises the first 15 amino acids of SEQ ID NO: 43.In one embodiment, the truncated CTP comprises the first 16 amino acidsof SEQ ID NO: 43. In one embodiment, the truncated CTP comprises thelast 14 amino acids of SEQ ID NO: 43.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is glycosylated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In one embodiment, the CTP sequence of thepresent invention comprises at least one glycosylation site. In oneembodiment, the CTP sequence of the present invention comprises 2glycosylation sites. In one embodiment, the CTP sequence of the presentinvention comprises 3 glycosylation sites. In one embodiment, the CTPsequence of the present invention comprises 4 glycosylation sites. Eachpossibility represents a separate embodiment of the present invention.

As provided herein, attachment of CTP sequence to both the amino andcarboxy termini of the EPO protein results in increased potency atstimulating erythropoiesis (FIGS. 3-5 and Table 6 of Example 4), ascompared to recombinant EPO and other combinations of EPO and CTP. Insome embodiments, an EPO attached to three CTP sequences does not impairbinding to its receptor as evidenced in Table 4 of Example 3, whichdemonstrates that EPO attached to three CTP sequences is equallyeffective at stimulating proliferation of TF-1 cells as wild-type EPO.

In some embodiments, “homology” according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution thereof, and biologically active polypeptidefragments thereof. In one embodiment the substitution variant comprisesa substitution of the glycine in position 104 of the erythropoietinamino acid sequence with a serine (SEQ ID NO: 22).

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 1 having additionally at least one CTP amino acid peptide on theN-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 1 having additionally at least one CTP amino acid peptide on theN-terminus and at least one additional CTP amino acid peptide on theC-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 2 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 3having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 4having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 5having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 6having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 22having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of anemia. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide a nucleic acid sequence asset forth in SEQ ID NO: 20 encoding an EPO peptide and one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide a nucleic acid sequence asset forth in SEQ ID NO: 21 encoding an EPO peptide and one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for the treatment of anemia.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for inhibitinganemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for inhibiting anemia.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for the treatment oftumor-associated anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for the treatment of tumor-associated anemia.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for inhibitingtumor-associated anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for inhibiting tumor-associated anemia.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides, as described herein, for the treatmentof tumor hypoxia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for the treatment of tumor hypoxia.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for the treatment ofchronic infections such as HIV, inflammatory bowel disease, or septicepisodes.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptides forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for inhibitingchronic infections such as HIV, inflammatory bowel disease, or septicepisodes.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CT-modified EPO peptidesdescribed herein, for inhibiting chronic infections such as HIV,inflammatory bowel disease, or septic episodes.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for the treatment offatigue syndrome following cancer chemotherapy.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for the treatment of fatigue syndrome following cancerchemotherapy.

In another embodiment, the methods of the present invention provide anyof the CTP-modified EPO peptides described herein, for improving stemcell engraftment.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for improving stem cell engraftment.

In another embodiment, the methods of the present invention provide anyof the CTP-modifieds EPO peptide described herein, for increasing thesurvival rate of a patient with aplastic anemia or myelodysplasticsyndrome.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified EPO peptidesdescribed herein, for increasing the survival rate of a patient withaplastic anemia or myelodysplastic syndrome.

In some embodiments, human growth hormone (hGH) is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequence to both the amino and carboxy termini of thehGH protein results in increased potency (FIG. 11). In some embodiments,the attachment of CTP sequence to both the amino and carboxy termini ofthe hGH protein results in prolonged in-vivo activity. In oneembodiment, CTP-hGH polypeptides of the present invention are set forthin SEQ ID NO: 39-41.

As provided herein, growth gain was demonstrated in hypophysectomizedrats (which have no growth hormone secretion) following injections ofCTP-hGH.

In one embodiment, the phrase “human growth hormone” (hGH) refers to apolypeptide, such as set forth in Genbank Accession No. P01241 (SEQ IDNO: 47), exhibiting hGH activity (i.e. stimulation of growth).

In another embodiment, “human growth hormone” (hGH) refers to apolypeptide, such as set forth in Genbank Accession No. P01241,exhibiting hGH activity (i.e. stimulation of growth). In anotherembodiment, “GH” of the present invention also refers to homologues. Inanother embodiment, a GH amino acid sequence of the methods andcompositions the present invention is at least 50% homologous to a GHsequence set forth herein as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In another embodiment, the percent homology is 60%. Inanother embodiment, the percent homology is 70%. In another embodiment,the percent homology is 80%. In another embodiment, the percent homologyis 90%. In another embodiment, the percent homology is at least 95%. Inanother embodiment, the percent homology is greater than 95%. Eachpossibility represents a separate embodiment of the present invention.

Exemplary CTP-GH polypeptides and CTP-hGH polypeptides of the presentinvention are set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO:41.

In another embodiment, the methods of the present invention provide agrowth hormone (GH) peptide having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth. In another embodiment,the methods of the present invention provide a GH peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for stimulating muscle growth. Inanother embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 23 having additionally at least one CTPamino acid peptide on the N-terminus and at least one CTP amino acidpeptide on the C-terminus for stimulating muscle growth. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 36 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth. In another embodiment,the methods of the present invention provide a GH peptide set forth inSEQ ID NO: 37 having additionally at least one CTP amino acid peptide onthe N-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 38, having additionally at least one CTP amino acid peptide onthe N-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 39 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 40 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 41 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 42 having additionally at least one CTP amino acid peptide on theN-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide modified by CTPsas provided herein for stimulating muscle growth.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for stimulating muscle growth. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forstimulating muscle growth. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for stimulating muscle growth.

In one embodiment, the present invention provides a method of reducingthe dosing frequency of a growth hormone in a subject, comprisingadministering to said subject a therapeutically effective amount of apolypeptide consisting of a growth hormone, one chorionic gonadotrophincarboxy terminal peptide (CTP) attached to the amino terminus of saidgrowth hormone, and two chorionic gonadotrophin CTPs attached to thecarboxy terminus of said growth hormone, and wherein said polypeptideoptionally consists of a signal peptide attached to the amino terminusof said one CTP, thereby reducing the dosing frequency of a growthhormone in a subject.

In another embodiment, the present invention provides a method ofimproving the area under the curve (AUC) of a growth hormone in asubject, comprising administering to said subject a therapeuticallyeffective amount of a polypeptide consisting of a growth hormone, onechorionic gonadotrophin carboxy terminal peptide (CTP) attached to theamino terminus of said growth hormone, and two chorionic gonadotrophinCTPs attached to the carboxy terminus of said growth hormone, andwherein said polypeptide optionally consists of a signal peptideattached to the amino terminus of said one CTP, thereby reducing thedosing frequency of a growth hormone in a subject.

In one embodiment, the present invention provides a method of treating asubject in need of GH therapy, comprising administering to said subjecta therapeutically effective amount of a polypeptide consisting of agrowth hormone, one chorionic gonadotrophin carboxy terminal peptide(CTP) attached to the amino terminus of said growth hormone, and twochorionic gonadotrophin CTPs attached to the carboxy terminus of saidgrowth hormone, and wherein said polypeptide optionally consists of asignal peptide attached to the amino terminus of said one CTP, therebyreducing the dosing frequency of a growth hormone in a subject.

In another embodiment, the present invention provides a method ofincreasing insulin-like growth factor (IGF-1) levels in a subject,comprising administering to said subject a therapeutically effectiveamount of a polypeptide consisting of a growth hormone, one chorionicgonadotrophin carboxy terminal peptide (CTP) attached to the aminoterminus of said growth hormone, and two chorionic gonadotrophin CTPsattached to the carboxy terminus of said growth hormone, and whereinsaid polypeptide optionally consists of a signal peptide attached to theamino terminus of said one CTP, thereby increasing insulin-like growthfactor (IGF-1) levels in a subject.

In another embodiment, the methods of the present invention provide anyof the CTP-modified GH peptides described herein, for stimulating bonegrowth.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified GH peptidesdescribed herein, for stimulating bone growth.

In another embodiment, conjugated growth hormones of this invention areused in the same manner as unmodified growth hormones. In anotherembodiment, conjugated growth hormones of this invention have anincreased circulating half-life and plasma residence time, decreasedclearance, and increased clinical activity in vivo. In anotherembodiment, due to the improved properties of the conjugated growthhormones as described herein, these conjugates are administered lessfrequently than unmodified growth hormones. In another embodiment,conjugated growth hormones as described herein are administered once aweek to once every two weeks. In another embodiment, conjugated growthhormones as described herein are administered once every two weeks toonce every three weeks. In another embodiment, conjugated growthhormones as described herein are administered once a day to three timesa week. In another embodiment, decreased frequency of administrationwill result in improved patient compliance leading to improved treatmentoutcomes, as well as improved patient quality of life. In anotherembodiment, compared to conventional conjugates of growth hormoneslinked to poly(ethylene glycol) it has been found that growth hormoneCTP conjugates having the molecular weight and linker structure of theconjugates of this invention have an improved potency, improvedstability, elevated AUC levels, enhanced circulating half-life. Inanother embodiment, compared to conventional conjugates of growthhormones linked to poly(ethylene glycol) it has been found that growthhormones having the molecular weight and linker structure of theconjugates of this invention have an improved potency, improvedstability, elevated AUC levels, enhanced circulating half-life. Inanother embodiment, a therapeutically effective amount of a conjugatedgrowth hormone is the amount of conjugate necessary for the in vivomeasurable expected biological activity. In another embodiment, a growthhormone utilized according to the teachings of the present inventionexhibits increased potency. In some embodiments, the attachment of CTPsequence to both the amino and carboxy termini of a growth hormoneresults in prolonged in-vivo activity.

In another embodiment, a therapeutically effective amount of aconjugated growth hormone is determined according to factors as theexact type of condition being treated, the condition of the patientbeing treated, as well as the other ingredients in the composition. Inanother embodiment, a therapeutically effective amount of a conjugatedgrowth hormone is 0.01 to 10 μg per kg body weight administered once aweek. In another embodiment, a therapeutically effective amount of aconjugated growth hormone is 0.1 to 1 μg per kg body weight,administered once a week. In another embodiment, a pharmaceuticalcomposition comprising a conjugated growth hormone is formulated atstrength effective for administration by various means to a humanpatient.

In another embodiment, the growth hormone is any growth hormone known toone of skill in the art. In another embodiment, the growth hormone is ahuman growth hormone. In another embodiment, the growth hormone is anon-human growth hormone. In another embodiment, the nucleotide sequenceand/or the amino acid sequence of a growth hormone is available in agene bank database. In another embodiment, the growth hormone is ahomologue. In another embodiment, a homologue also refers to a deletion,insertion, or substitution variant, including an amino acidsubstitution, thereof and biologically active polypeptide fragmentsthereof.

In another embodiment, the growth hormone is variant of hGH missingexons 2, 3, 4, or any combination thereof. In another embodiment, thegrowth hormone comprises a signal peptide. In another embodiment, thegrowth hormone comprises a signal cleavage site. In another embodiment,polypeptides comprising GH modified by CTPs of the present inventioncomprise recombinant GH.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for maintaining muscle quality.

In another embodiment, the methods of the present invention provide a GHof the present invention for maintaining bone quality.

In another embodiment, the methods of the present invention provide aGH-CTP nucleic acid sequence of the present invention for maintainingbone quality.

In another embodiment, the methods of the present invention provide anyof the CTP-modified GH peptides described herein, for treating a wastingdisease.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified GH peptidesdescribed herein, for treating a wasting disease.

In another embodiment, the methods of the present invention provide anyof the CTP-modified GH peptides described herein, for increasing cardiacfunction.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified GH peptidesdescribed herein, for increasing cardiac function.

In another embodiment, the methods of the present invention provide a GHpeptides described herein, for increasing lipolysis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding any of the CTP-modified GH peptidesdescribed herein, for increasing lipolysis.

In another embodiment, the methods of the present invention provide anyof the CTP-modified GH peptides described herein, for improving fluidbalance.

In another embodiment, a growth hormone of the invention comprises thegene bank amino acid deposited sequence under accession no. AAA72260. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. AAK69708. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. CAA01435. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. CAA01329. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. CAA00380. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. AAA72555. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. NP_000506.2. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. NP_072053.1. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. NP_072054.1. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. NP_072055.1. Inanother embodiment, a growth hormone of the invention comprises the genebank amino acid deposited sequence under accession no. NP_072056.1.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving fluid balance. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving fluid balance. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving fluid balance.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for treating osteoporosis. In another embodiment, the methodsof the present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 39 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 40 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 41 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide modified by CTPs, as provided herein, fortreating osteoporosis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for treating osteoporosis. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for treating osteoporosis. In another embodiment, the methodsof the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a nucleic acid of SEQ ID NO: 46 encoding a GH peptideand one CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for treating osteoporosis.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting osteoporosis. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide modified by CTPs, as providedherein, for inhibiting osteoporosis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for inhibiting osteoporosis. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting osteoporosis. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting osteoporosis.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for improving exercise capacity.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving lung function. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 40 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 41 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 42 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide modified by CTPs as provided herein for improvinglung function.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving lung function. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving lung function. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving lung function.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving immunity. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity. In another embodiment, the methods ofthe present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving immunity. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for improving immunity.In another embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 40 for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 41 for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 42 having additionally at least one CTP aminoacid peptide on the N-terminus for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptidemodified by CTPs, as provided herein, for improving immunity.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving immunity. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity. In another embodiment, the methods ofthe present invention provide a nucleic acid of SEQ ID NO: 45 encoding aGH peptide comprising one CTP amino acid peptide on the N-terminus andtwo CTP amino acid peptides on the C-terminus for improving immunity. Inanother embodiment, the methods of the present invention provide anucleic acid of SEQ ID NO: 46 encoding a GH peptide and one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for regrowing vital organs. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for regrowingvital organs. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide modified by CTPs, as providedherein, for regrowing vital organs.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for regrowing vital organs. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for regrowing vital organs. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for regrowing vital organs.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for increasing sense of well-being.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for restoring REM sleep. In another embodiment, the methodsof the present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for restoring REM sleep.In another embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 40 for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 41 for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 42 having additionally at least one CTP aminoacid peptide on the N-terminus for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptidemodified by CTPs, as provided herein, for restoring REM sleep.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for restoring REM sleep. In another embodiment, the methodsof the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a nucleic acid of SEQ ID NO: 46 encoding a GH peptideand one CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for restoring REM sleep.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH protein as described herein. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding polypeptide comprising hGH modified byCTPs for stimulating muscle growth, increasing cardiac function,stimulating bone growth, maintaining muscle integrity, balancing musclemetabolism, inducing muscle buildup, inducing de-novo muscle build-up,enhancing bone load, treating symptoms associated with osteoporosis,treating a wasting disease, increasing lipolysis, improving fluidbalance, treating osteoporosis, improving lung function, improvingimmunity, regrowing a vital organ, increasing sense of well-being,restoring REM sleep, or any combination thereof.

In some embodiments, “homology” according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution, thereof and biologically active polypeptidefragments thereof. In one embodiment the substitution variant is one inwhich the glutamine in position 65 of hGH is substituted by a valine[Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].

In another embodiment, the nucleic acid molecule encoding a growthhormone as described herein encodes any amino acid sequence of a growthhormone known to one of skill in the art. In another embodiment, thenucleic acid molecule encoding a growth hormone as described hereinencodes an hGH. In another embodiment, the nucleic acid moleculeencoding a growth hormone comprises the gene bank nucleic acid depositedsequence under accession no. NM_000515.3. In another embodiment, thenucleic acid molecule encoding a growth hormone comprises the gene banknucleic acid deposited sequence under accession no. NM_022559.2. Inanother embodiment, the nucleic acid molecule encoding a growth hormonecomprises the gene bank nucleic acid deposited sequence under accessionno. NM_022560.2. In another embodiment, the nucleic acid moleculeencoding a growth hormone comprises the gene bank nucleic acid depositedsequence under accession no. NM_022561.2. In another embodiment, thenucleic acid molecule encoding a growth hormone comprises the gene banknucleic acid deposited sequence under accession no. NM_022562.2.

In some embodiments, interferon is utilized according to the teachingsof the present invention. In some embodiments, the attachment of CTPsequence to both the amino and carboxy termini of the interferon proteinresults in increased potency. In some embodiments, the attachment of CTPsequence to both the amino and carboxy termini of the interferon proteinresults in prolonged in-vivo activity.

In one embodiment, “interferon” refers to the mammalian interferonpolypeptide Type I. In one embodiment, “interferon” refers to themammalian interferon polypeptide Type II. In some embodiments,additional suitable interferon polypeptides as known to those ofordinary skill in the art are utilized. In some embodiments, theinterferon is alpha-interferon. In some embodiments, the interferon isbeta-interferon. In some embodiments, the interferon isgamma-interferon. In some embodiments, the interferon isomega-interferon. In some embodiments, the interferon is a subspeciesinterferon. In one embodiment, the subspecies of interferon (IFN) isIFN-α2a. In one embodiment, the subspecies of interferon (IFN) isIFN-α2b. In one embodiment, the subspecies of interferon (IFN) isIFN-β1a. In one embodiment, the interferon (IFN) subspecies is IFN-β1b.

In one embodiment, interferon of the present invention exhibitsinterferon activity, such as antiviral or antiproliferative activity. Insome embodiments, GenBank=Accession Numbers of non-limiting examples ofinterferons are listed in Table 1 below.

In one embodiment, an interferon of the present invention also refers tohomologues. In one embodiment, an interferon amino acid sequence of thepresent invention is at least 50% homologous to an interferon sequencedisclosed herein. In one embodiment, an interferon amino acid sequenceof the present invention is at least 60% homologous an interferonsequence disclosed herein. In one embodiment, an interferon amino acidsequence of the present invention is at least 70% homologous aninterferon sequence disclosed herein. In one embodiment, an interferonamino acid sequence of the present invention is at least 80% homologousto an interferon sequence disclosed herein). In one embodiment,interferon amino acid sequence of the present invention is at least 90%homologous to an interferon sequence disclosed herein. In oneembodiment, an interferon amino acid sequence of the present inventionis at least 95% homologous an interferon sequence disclosed herein. Insome embodiments, homology according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution, thereof and biologically active polypeptidefragments thereof. In one embodiment the cysteine in position 17 ofinterferon β is substituted by a serine (SEQ ID NO: 24).

Table 1 below lists examples of interferons with their respective NCBIsequence numbers

TABLE 1 Interferon name NCBI sequence number interferon, α 1 NP_076918.1interferon, α 10 NP_002162.1 interferon, α13 NP_008831.2 interferon, α14NP_002163.1 interferon, α16 NP_002164.1 interferon, α17 NP_067091.1interferon, α2 NP_000596.2 interferon, α21 NP_002166.1 interferon, α4NP_066546.1 interferon, α5 NP_002160.1 interferon, α6 NP_066282.1interferon, α7 NP_066401.2 interferon, α8 NP_002161.2 interferon, β 1NP_002167.1 interferon, ε1 NP_795372.1 interferon, γ NP_000610.2interferon, ε NP_064509.1 interferon, Ω1 NP_002168.1

In another embodiment, the methods of the present invention provide aninterferon beta 1 peptide having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for treating or inhibiting multiple sclerosis. Inanother embodiment, the methods of the present invention provide aninterferon beta 1 peptide having additionally one CTP amino acid peptideon the N-terminus and two CTP amino acid peptides on the C-terminus fortreating or inhibiting multiple sclerosis. In another embodiment, themethods of the present invention provide an interferon beta 1 peptideset forth in SEQ ID NO: 24 having additionally at least one CTP aminoacid peptide on the N-terminus and one CTP amino acid peptide on theC-terminus for treating or inhibiting multiple sclerosis. In anotherembodiment, the methods of the present invention provide an interferonbeta 1 peptide set forth in SEQ ID NO: 24 having additionally on theN-terminus the signal peptide of SEQ ID NO: 26 and at least one CTPamino acid peptide on the N-terminus of SEQ ID NO: 26 and at least oneCTP amino acid peptide on the C-terminus of SEQ ID NO: 24 for treatingor inhibiting multiple sclerosis.

In some embodiments, glucagon-like peptide-1 is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequences to both the amino and carboxy termini of a“glucagon-like peptide-1” results in increased potency. In someembodiments, the attachment of CTP sequences to both the amino andcarboxy termini of a peptide results in prolonged in-vivo activity. Insome embodiments, the attachment of CTP sequences to both the amino andcarboxy termini of the glucagon-like peptide-results in prolongedin-vivo activity.

In one embodiment, “glucagon-like peptide-1” (GLP-1) refers to amammalian polypeptide. In one embodiment, “glucagon-like peptide-1”(GLP-1) refers to a human polypeptide. In some embodiments, GLP-1 iscleaved from the glucagon preproprotein (Genbank ID No. NP002045) thathas the ability to bind to the GLP-1 receptor and initiate a signaltransduction pathway resulting in insulinotropic activity. In oneembodiment, “insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and decreased plasma glucose levels. Insome embodiments, GLP-1 polypeptides include, but are not limited tothose described in U.S. Pat. No. 5,118,666; which is incorporated byreference herein.

In one embodiment, “GLP-1” refers to a polypeptide, such as set forth insequences set forth in SEQ ID NO: 25 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 of the present invention alsorefers to a GLP-1 homologue. In one embodiment, a GLP-1 amino acidsequence of the present invention is at least 50% homologous to GLP-1sequences set forth in SEQ ID NO: 25 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, aGLP-1 amino acid sequence of the presentinvention is at least 60% homologous to GLP-1 sequences set forth in SEQID NO: 25 as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters. In oneembodiment, aGLP-1 amino acid sequence of the present invention is atleast 70% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, a GLP-1amino acid sequence of the present invention is at least 80% homologousto GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters. In one embodiment, a GLP-1 amino acid sequenceof the present invention is at least 90% homologous to GLP-1 sequencesset forth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 95% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters.

In another embodiment, the methods of the present invention provide aGLP-1 peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor treating or inhibiting type II diabetes. In another embodiment, themethods of the present invention provide a GLP-1 peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for treating or inhibiting type IIdiabetes. In another embodiment, the methods of the present inventionprovide a GLP-1 peptide set forth in SEQ ID NO: 25 having additionallyat least one CTP amino acid peptide on the N-terminus and at least oneCTP amino acid peptide on the C-terminus for treating or inhibiting tuneII diabetes.

In one embodiment, the homologue also refers to a deletion, insertion,or substitution variant, including an amino acid substitution, thereofand biologically active polypeptide fragments thereof.

In one embodiment the polypeptide sequence of interest is an EPO. In oneembodiment the polypeptide sequence of interest is an interferon. Inanother embodiment the polypeptide sequence of interest is an hGH. Inanother embodiment the polypeptide sequence of interest is a GLP-1. Inanother embodiment the polypeptide sequence of interest is an insulin.In another embodiment the polypeptide sequence of interest isenkephalin. In another embodiment the polypeptide sequence of interestis an ACTH. In another embodiment the polypeptide sequence of interestis a glucagon. In another embodiment the polypeptide sequence ofinterest is an insulin-like growth factor. In another embodiment thepolypeptide sequence of interest is an epidermal growth factor. Inanother embodiment the polypeptide sequence of interest is an acidic orbasic fibroblast growth factor. In another embodiment the polypeptidesequence of interest is a platelet-derived growth factor. In anotherembodiment the polypeptide sequence of interest is a granulocyte-CSF. Inanother embodiment the polypeptide sequence of interest is amacrophage-CSF. In another embodiment the polypeptide sequence ofinterest is an IL-2. In another embodiment the polypeptide sequence ofinterest is an IL-3. In another embodiment the polypeptide sequence ofinterest is a tumor necrosis factor. In another embodiment thepolypeptide sequence of interest is an LHRH. In another embodiment thepolypeptide sequence of interest is an LHRH analog. In anotherembodiment the polypeptide sequence of interest is a somatostatin. Inanother embodiment the polypeptide sequence of interest is a growthhormone releasing factor. In another embodiment the polypeptide sequenceof interest is an endorphin. In another embodiment the polypeptidesequence of interest is an alveolar surfactant protein. In anotherembodiment the polypeptide sequence of interest is a natriuretic factor.In another embodiment the polypeptide sequence of interest is anadhesion. In another embodiment the polypeptide sequence of interest isan angiostatin. In another embodiment the polypeptide sequence ofinterest is an endostatin. In another embodiment the polypeptidesequence of interest is a receptor peptide. In another embodiment thepolypeptide sequence of interest is a receptor binding ligand. Inanother embodiment the polypeptide sequence of interest is an antibody.In another embodiment the polypeptide sequence of interest is anantibody fragment. In another embodiment the polypeptide sequence ofinterest is a peptide or a protein including any modified form.

In another embodiment, the peptide of the invention comprises a peptideof interest having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus. Inanother embodiment, the peptide of interest having additionally at leastone CTP amino acid peptide on the N-terminus and one CTP amino acidpeptide on the C-terminus comprises a protein selected from thefollowing list: insulin, Albutein/albumin, Activase® Alteplase/tPA,adenosine deaminase, immune globulin, glucocerebrosidase,Leukine-sargramostim/GM-CSF, G-CSF, Venoglobulin-S/IgG, Proleukinaldesleukin, DNase, Factor VIII, Helixate, L-asparaginase, WinRho SDF RhI, Retavase retaplase/tPA, Factor IX, FSH, globulin, fibrin,interleukin-11, becaplermin/PDGF, lepirudin/herudin, TNF, Thymoglobulin,Factor VIIa, interferon alpha-2a, interferon alfa n-1, interferonalfa-N3, interferon beta-1b, interferon gamma-1b, Interleukin-2, HGH, ormonoclonal antibodies.

In another embodiment, the methods of the present invention provideinsulin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of diabetes.

In another embodiment, the methods of the present invention providealbumin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of hypovolemic shock, hemodialysis or cardiopulmonary bypass.

In another embodiment, the methods of the present invention provideActivase-altiplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction, acutemassive pulmonary embolism, or ischemic stroke.

In another embodiment, the methods of the present invention provideadenosine deaminase having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of severe combined immunodeficiencydisease.

In another embodiment, the methods of the present invention provideimmune globulin having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of transplant recipients.

In another embodiment, the methods of the present invention provide aCMV immune globulin. In another embodiment, the methods of the presentinvention provide glucocerebrosidase having additionally at least oneCTP amino acid peptide on the N-terminus and one CTP amino acid peptideon the C-terminus for the treatment of Gaucher disease.

In another embodiment, the methods of the present invention provideLeukine-sargramostim/GM-CSF having additionally at least one CTP aminoacid peptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the Stimulation of hematopoietic progenitor cells.

In another embodiment, the methods of the present invention provideG-CSF having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Neutropenia. In another embodiment, the methods of thepresent invention provide Venoglobulin-S/IgG having additionally atleast one CTP amino acid peptide on the N-terminus and one CTP aminoacid peptide on the C-terminus for the treatment of Immunodeficiencydiseases.

In another embodiment, the methods of the present invention provideProleukin (aldesleukin) having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of renal carcinoma or metastatic melanoma.

In another embodiment, the methods of the present invention provideDNase having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Cystic fibrosis.

In another embodiment, the methods of the present invention provideFactor VIII having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention provideHelixate having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention provideL-asparaginase having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of acute lymphoblastic leukemia.

In another embodiment, the methods of the present invention provideWinRho SDF Rh IgG having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of Rh isoimmunization and immunethrombocytopenic purpura.

In another embodiment, the methods of the present invention provideRetavase retaplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction.

In another embodiment, the methods of the present invention provideFactor IX having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of Hemophilia B.

In another embodiment, the methods of the present invention provide FSHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forstimulation of ovulation during assisted reproduction.

In another embodiment, the methods of the present invention provideglobulin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe prevention of respiratory syncytial virus disease.

In another embodiment, the methods of the present invention providefibrin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forwound management and hemostasis. In another embodiment, the methods ofthe present invention provide interleukin-11 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for chemotherapy-inducedthrombocytopenia.

In another embodiment, the methods of the present invention providebecaplermin/PDGF having additionally at least one CTP amino acid peptideon the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of diabetic foot ulcers.

In another embodiment, the methods of the present invention providelepirudin/herudin having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for anticoagulation in heparin-induced thrombocytopenia.

In another embodiment, the methods of the present invention providesoluble TNF having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of rheumatoid arthritis.

In another embodiment, the methods of the present invention provideThymoglobulin having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of organ transplant rejection disease.

In another embodiment, the methods of the present invention provideFactor VIIa having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of hemophilia.

In another embodiment, the methods of the present invention provideinterferon alpha-2a having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of hairy cell leukemia and AIDS-relatedKaposi's sarcoma.

In another embodiment, the methods of the present invention provideinterferon alpha-2b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of Hairy cell leukemia, genital warts,AIDS-related Kaposi's sarcoma, Hepatitis C, Hepatitis B, malignantmelanoma, and follicular lymphoma.

In another embodiment, the methods of the present invention provideinterferon alfa-N3 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of genital warts.

In another embodiment, the methods of the present invention provideinterferon gamma-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of chronic granulomatous disease.

In another embodiment, the methods of the present invention provideinterferon alfa n-1 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of Hepatitis C infection.

In another embodiment, the methods of the present invention provideInterleukin-2 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of renal carcinoma and metastatic melanoma.

In another embodiment, the methods of the present invention provideinterferon beta-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of multiple sclerosis.

In another embodiment, the methods of the present invention provide hGHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of wasting disease, AIDS, cachexia, or hGH deficiency.

In another embodiment, the methods of the present invention provide anOKT3 monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for organ transplant.

In another embodiment, the methods of the present invention provide aReo monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for prevention of complications from coronary interventionand angioplasty.

In another embodiment, the methods of the present invention provide amonoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for treating colorectal cancer, Non-Hodgkin's lymphoma,kidney transplant rejection, metastatic breast cancer, or the preventionof respiratory syncytial virus disease.

In one embodiment, the invention is employed in veterinary medicine. Inone embodiment, the present invention provides treatment of domesticatedmammals which are maintained as human companions (e.g., dogs, cats,horses), which have significant commercial value (e.g., dairy cows. beefcattle, sporting animals), which have significant scientific value(e.g., captive or free specimens of endangered species), or whichotherwise have value.

In one embodiment, polypeptides, antibodies, or polynucleotides of thepresent invention are administered to an animal (e.g., mouse, rat,rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat,horse, cow, sheep, dog, cat, non-human primate, and human. In oneembodiment, the recited applications have uses in a wide variety ofhosts. In some embodiments, such hosts include, but are not limited to,human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, ornon-human primate.

In one embodiment, farm animals are treated by the methods of thepresent invention. In one embodiment, farm animals include pigs, cattle,dairy cows, horses, goats, sheep, chickens, turkeys, geese, ducks andrelated species. In one embodiment, laboratory animals are treated bythe methods of the present invention. In one embodiment, laboratoryanimals include rats, mice, guinea pigs, rabbits, goats, monkeys, dogs,cats and others. In one embodiment, zoo animals are treated by themethods of the present invention. In one embodiment, zoo animals includeall vertebrate animals kept in zoos. In one embodiment, aquatic animalsare treated by the methods of the present invention. In one embodiment,aquatic animals include fish, eels, turtles, seals, penguins, sharks,whales, and related species. In one embodiment, domesticated animals aretreated by the methods of the present invention. In one embodiment,domesticated animals include any pet, such as cats and dogs, or animalthat is kept by humans, e.g., horses, cattle, pigs, goats, rabbits,chickens, turkeys, geese, ducks and the like.

According to the present invention the term pigs includes pigs, piglets,hogs, gilts, barrows, boars and sows. In another embodiment, “cattle”refers to calves, cows, dairy cows, heifers, steers and bulls.

In one embodiment, bovine growth hormone is utilized by the methods ofthe present invention. In one embodiment, artificial bovine growthhormone is utilized by the methods of the present invention. In oneembodiment, the artificial bovine growth hormone has a sequence setforth in NCBI sequence ID number AAA72262. In another embodiment, theartificial bovine growth hormone is any other artificial bovine growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, sheep growth hormone is utilized by the methods ofthe present invention. In one embodiment, sheep growth hormone has asequence set forth in NCBI sequence ID number NP_001009315. In anotherembodiment, the sheep growth hormone is any other sheep growth hormoneknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, horse growth hormone is utilized by the methods ofthe present invention. In one embodiment, horse growth hormone has asequence set forth in NCBI sequence ID number AAA21027. In anotherembodiment, the horse growth hormone is any other horse growth hormoneknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, chicken growth hormone is utilized by the methods ofthe present invention. In one embodiment, chicken growth hormone has asequence set forth in NCBI sequence ID number CAA3561. In anotherembodiment, the chicken growth hormone is any other chicken growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, murine growth hormone is utilized by the methods ofthe present invention. In one embodiment, the murine growth hormone hasa sequence set forth in NCBI sequence ID number NP_032143. In anotherembodiment, the murine growth hormone is any other murine growth hormoneknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, tilapia growth hormone is utilized by the methods ofthe present invention. In one embodiment, the tilapia growth hormone hasa sequence set forth in NCBI sequence ID number CAA00818. In anotherembodiment, the tilapia growth hormone is any other tilapia growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, bovine EPO is utilized by the methods of the presentinvention. In one embodiment, artificial bovine growth hormone isutilized by the methods of the present invention. In one embodiment,artificial bovine growth hormone has a sequence set forth in NCBIsequence ID number NP_776334. In another embodiment, the bovine EPO isany other bovine EPO known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, pig EPO is utilized by the methods of the presentinvention. In one embodiment, pig EPO has a sequence set forth in NCBIsequence ID number NP_999299. In another embodiment, the pig EPO is anyother pig EPO known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, sheep EPO is utilized by the methods of the presentinvention. In one embodiment, sheep growth hormone has a sequence setforth in NCBI sequence ID number NP_001019908. In another embodiment,the sheep growth hormone is any other sheep growth hormone known in theart. Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, murine EPO is utilized by the methods of the presentinvention. In one embodiment, the murine growth hormone has a sequenceset forth in NCBI sequence ID number CAA72707. In another embodiment,the murine growth hormone is any other murine growth hormone known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, bovine GLP-1 is utilized by the methods of thepresent invention. In one embodiment, bovine GLP-1 has a sequence setforth in NCBI sequence ID number P01272. In another embodiment, thebovine GLP-1 is any other bovine GLP-1 known in the art. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, sheep GLP-1 is utilized by the methods of the presentinvention. In one embodiment, sheep GLP-1 has a sequence set forth inNCBI sequence ID number Q8MJ25. In another embodiment, the sheep GLP-1is any other sheep GLP-1 known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, pig GLP-1 is utilized by the methods of the presentinvention. In one embodiment, chicken GLP-1 has a sequence set forth inNCBI sequence ID number P01274. In another embodiment, the chicken GLP-1is any other chicken GLP-1 known in the art. Each possibility representsa separate embodiment of the present invention.

In one embodiment, murine GLP-1 is utilized by the methods of thepresent invention. In one embodiment, the murine GLP-1 has a sequenceset forth in NCBI sequence ID number NP_032127. In another embodiment,the murine GLP-1 is any other murine GLP-1 known in the art. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, bovine interferon alpha is utilized by the methods ofthe present invention. In one embodiment, bovine interferon alpha has asequence set forth in NCBI sequence ID number ABD57311. In anotherembodiment, the bovine interferon alpha is any other bovine interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, sheep interferon alpha is utilized by the methods ofthe present invention. In one embodiment, sheep interferon alpha has asequence set forth in NCBI sequence ID number CAA41790. In anotherembodiment, the sheep interferon alpha is any other sheep interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, pig interferon alpha is utilized by the methods ofthe present invention. In one embodiment, chicken interferon alpha has asequence set forth in NCBI sequence ID number AAP92118. In anotherembodiment, the pig interferon alpha is any other pig interferon alphaknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, murine interferon alpha is utilized by the methods ofthe present invention. In one embodiment, the murine interferon alphahas a sequence set forth in NCBI sequence ID number AAA37886. In anotherembodiment, the murine interferon alpha is any other murine interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In some embodiments, the CTP sequences modification is advantageous inpermitting lower dosages to be used.

In some embodiments, “polypeptide” as used herein encompasses nativepolypeptides (either degradation products, synthetically synthesizedpolypeptides or recombinant polypeptides) and peptidomimetics(typically, synthetic polypeptides), as well as peptoids andsemipeptoids which are polypeptide analogs, which have, in someembodiments, modifications rendering the polypeptides even more stablewhile in a body or more capable of penetrating into cells.

In some embodiments, modifications include, but are not limited to Nterminus modification, C terminus modification, polypeptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinbelow.

In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptideare substituted. In some embodiments, the polypeptide bonds aresubstituted by N-methylated bonds (—N(CH3)-CO—). In some embodiments,the polypeptide bonds are substituted by ester bonds(—C(R)H—C—O—O—C(R)—N—). In some embodiments, the polypeptide bonds aresubstituted by ketomethylen bonds (—CO—CH2-). In some embodiments, thepolypeptide bonds are substituted by α-aza bonds (—NH—N(R)—CO—), whereinR is any alkyl, e.g., methyl, carba bonds (—CH2-NH—). In someembodiments, the polypeptide bonds are substituted by hydroxyethylenebonds (—CH(OH)—CH2-). In some embodiments, the polypeptide bonds aresubstituted by thioamide bonds (—CS—NH—). In some embodiments, thepolypeptide bonds are substituted by olefinic double bonds (—CH═CH—). Insome embodiments, the polypeptide bonds are substituted by retro amidebonds (—NH—CO—). In some embodiments, the polypeptide bonds aresubstituted by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the“normal” side chain, naturally presented on the carbon atom. In someembodiments, these modifications occur at any of the bonds along thepolypeptide chain and even at several (2-3 bonds) at the same time.

In some embodiments, natural aromatic amino acids of the polypeptidesuch as Trp, Tyr and Phe, be substituted for synthetic non-natural acidsuch as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylatedderivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. Insome embodiments, the polypeptides of the present invention include oneor more modified amino acid or one or more non-amino acid monomers (e.g.fatty acid, complex carbohydrates etc).

In one embodiment, “amino acid” or “amino acid” is understood to includethe 20 naturally occurring amino acid; those amino acid often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment,“amino acid” includes both D- and L-amino acid.

In some embodiments, the polypeptides of the present invention areutilized in therapeutics which requires the polypeptides to be in asoluble form. In some embodiments, the polypeptides of the presentinvention include one or more non-natural or natural polar amino acid,including but not limited to serine and threonine which are capable ofincreasing polypeptide solubility due to their hydroxyl-containing sidechain.

In some embodiments, the polypeptides of the present invention areutilized in a linear form, although it will be appreciated by oneskilled in the art that in cases where cyclicization does not severelyinterfere with polypeptide characteristics, cyclic forms of thepolypeptide can also be utilized.

In some embodiments, the polypeptides of the present invention arebiochemically synthesized such as by using standard solid phasetechniques. In some embodiments, these biochemical methods includeexclusive solid phase synthesis, partial solid phase synthesis, fragmentcondensation, or classical solution synthesis. In some embodiments,these methods are used when the polypeptide is relatively short (about5-15 kDa) and/or when it cannot be produced by recombinant techniques(i.e., not encoded by a nucleic acid sequence) and therefore involvesdifferent chemistry.

In some embodiments, solid phase polypeptide synthesis procedures arewell known to one skilled in the art and further described by JohnMorrow Stewart and Janis Dillaha Young, Solid Phase PolypeptideSyntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments,synthetic polypeptides are purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing by methods known to oneskilled in the art.

In some embodiments, recombinant protein techniques are used to generatethe polypeptides of the present invention. In some embodiments,recombinant protein techniques are used for generation of relativelylong polypeptides (e.g., longer than 18-25 amino acid). In someembodiments, recombinant protein techniques are used for the generationof large amounts of the polypeptide of the present invention. In someembodiments, recombinant techniques are described by Bitter et al.,(1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

In one embodiment, a polypeptide of the present invention is synthesizedusing a polynucleotide encoding a polypeptide of the present invention.In some embodiments, the polynucleotide encoding a polypeptide of thepresent invention is ligated into an expression vector, comprising atranscriptional control of a cis-regulatory sequence (e.g., promotersequence). In some embodiments, the cis-regulatory sequence is suitablefor directing constitutive expression of the polypeptide of the presentinvention. In some embodiments, the cis-regulatory sequence is suitablefor directing tissue specific expression of the polypeptide of thepresent invention. In some embodiments, the cis-regulatory sequence issuitable for directing inducible expression of the polypeptide of thepresent invention.

In some embodiments, polynucleotides which express the polypeptides ofthe present invention are as set forth in SEQ ID NOs: 20, 21, 44, 45 and46.

In some embodiment, tissue-specific promoters suitable for use with thepresent invention include sequences which are functional in specificcell population, example include, but are not limited to promoters suchas albumin that is liver specific [Pinkert et al., (1987) Genes Dev.1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv.Immunol. 43:235-275]; in particular promoters of T-cell receptors[Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerjiet al. (1983) Cell 33729-740], neuron-specific promoters such as theneurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985)Science 230:912-916] or mammary gland-specific promoters such as themilk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Inducible promoters suitable for use with thepresent invention include for example the tetracycline-induciblepromoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase “a polynucleotide” refers to a single ordouble stranded nucleic acid sequence which be isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the polypeptide of the presentinvention, as well as some intronic sequences interposing there between.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences include cisacting expression regulatory elements.

In one embodiment, the polynucleotides of the present invention furthercomprise a signal sequence encoding a signal peptide for the secretionof the polypeptides of the present invention. In some embodiments,signal sequences include, but are not limited to the endogenous signalsequence for EPO as set forth in SEQ ID NO: 19 or the endogenous signalsequence for IFN-β1 as set forth in SEQ ID NO: 26. In anotherembodiment, the signal sequence is N-terminal to the CTP sequence thatis in turn N-terminal to the polypeptide sequence of interest; e.g. thesequence is (a) signal sequence-(b) CTP-(c) sequence of interest-(d)optionally 1 or more additional CTP sequences. In another embodiment, 1or more CTP sequences is inserted between the signal sequence of apolypeptide sequence of interest and the polypeptide sequence ofinterest itself, thus interrupting the wild-type sequence of interest.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the growth hormone further comprises a signalpeptide. In some embodiments, signal sequences include, but are notlimited to the endogenous signal sequence. In some embodiments, signalsequences include, but are not limited to the endogenous signal sequenceof any known growth hormone or growth hormones. In another embodiment,the polypeptides and methods of the present invention provide a growthhormone having additionally a signal peptide of comprising the followingamino acid sequence: MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 49).

In one embodiment, following expression and secretion, the signalpeptides are cleaved from the precursor proteins resulting in the matureproteins. For example, in the polypeptide set forth in SEQ ID NO: 3,described herein and diagramed in FIG. 1C, amino acids 1-27 (SEQ ID NO:19), presented above, represent the amino acid sequence of the signalpeptide sequence, and amino acids 28-277 of SEQ ID NO: 3,SSSSKAPPPSLPSPSRLPGPSDTPILPQAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 51), represent the mature CTP-modifiedEPO lacking the signal peptide. In one embodiment, the amino acidsequence of a CTP-modified EPO having the structure CTP-EPO-CTP-CTPwithout the signal peptide is set forth in SEQ ID NO: 51.

In some embodiments, polynucleotides of the present invention areprepared using PCR techniques as described in Example 1, or any othermethod or procedure known to one skilled in the art. In someembodiments, the procedure involves the legation of two different DNAsequences (See, for example, “Current Protocols in Molecular Biology”,eds. Ausubel et al., John Wiley & Sons, 1992).

In one embodiment, polynucleotides of the present invention are insertedinto expression vectors (i.e., a nucleic acid construct) to enableexpression of the recombinant polypeptide. In one embodiment, theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes. In one embodiment, the expression vector of the presentinvention includes additional sequences which render this vectorsuitable for replication and integration in eukaryotes. In oneembodiment, the expression vector of the present invention includes ashuttle vector which renders this vector suitable for replication andintegration in both prokaryotes and eukaryotes. In some embodiments,cloning vectors comprise transcription and translation initiationsequences (e.g., promoters, enhancer) and transcription and translationterminators (e.g., polyadenylation signals).

In one embodiment, a variety of prokaryotic or eukaryotic cells can beused as host-expression systems to express the polypeptides of thepresent invention. In some embodiments, these include, but are notlimited to, microorganisms, such as bacteria transformed with arecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvector containing the polypeptide coding sequence; yeast transformedwith recombinant yeast expression vectors containing the polypeptidecoding sequence; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors,such as Ti plasmid, containing the polypeptide coding sequence.

In some embodiments, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express thepolypeptide of the present invention. In one embodiment, the expressionvector used to express polynucleotides of the present invention inmammalian cells is pCI-DHFR vector comprising a CMV promoter and aneomycin resistance gene. Construction of the pCI-dhfr vector isdescribed, according to one embodiment, in Example 1.

In some embodiments, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the polypeptide expressed. In one embodiment,large quantities of polypeptide are desired. In one embodiment, vectorsthat direct the expression of high levels of the protein product,possibly as a fusion with a hydrophobic signal sequence, which directsthe expressed product into the periplasm of the bacteria or the culturemedium where the protein product is readily purified are desired. In oneembodiment, certain fusion protein engineered with a specific cleavagesite to aid in recovery of the polypeptide. In one embodiment, vectorsadaptable to such manipulation include, but are not limited to, the pETseries of E. coli expression vectors [Studier et al., Methods inEnzymol. 185:60-89 (1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447. In anotherembodiment, vectors which promote integration of foreign DNA sequencesinto the yeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric polypeptide.

In some embodiments, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In some embodiments, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used by the presentinvention. SV40 vectors include pSVT7 and pMT2. In some embodiments,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p2O5. Otherexemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In some embodiments, recombinant viral vectors are useful for in vivoexpression of the polypeptides of the present invention since they offeradvantages such as lateral infection and targeting specificity. In oneembodiment, lateral infection is inherent in the life cycle of, forexample, retrovirus and is the process by which a single infected cellproduces many progeny virions that bud off and infect neighboring cells.In one embodiment, the result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. In one embodiment, viral vectors are produced that are unableto spread laterally. In one embodiment, this characteristic can beuseful if the desired purpose is to introduce a specified gene into onlya localized number of targeted cells.

In one embodiment, various methods can be used to introduce theexpression vector of the present invention into cells. Such methods aregenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992), inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press,Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

In some embodiments, introduction of nucleic acid by viral infectionoffers several advantages over other methods such as lipofection andelectroporation, since higher transfection efficiency can be obtaineddue to the infectious nature of viruses.

In one embodiment, it will be appreciated that the polypeptides of thepresent invention can also be expressed from a nucleic acid constructadministered to the individual employing any suitable mode ofadministration, described hereinabove (i.e., in-vivo gene therapy). Inone embodiment, the nucleic acid construct is introduced into a suitablecell via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.) and an expression systemas needed and then the modified cells are expanded in culture andreturned to the individual (i.e., ex-vivo gene therapy).

In one embodiment, in vivo gene therapy using EPO has been attempted inanimal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798],primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has provensuccessful in human clinical trials for patients with chronic renalfailure [Lippin et al Blood 2005, 106, Number 7].

In one embodiment, plant expression vectors are used. In one embodiment,the expression of a polypeptide coding sequence is driven by a number ofpromoters. In some embodiments, viral promoters such as the 35S RNA and19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)],or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311(1987)] are used. In another embodiment, plant promoters are used suchas, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] orheat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley etal., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructsare introduced into plant cells using Ti plasmid, Ri plasmid, plantviral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

Various methods, in some embodiments, can be used to introduce theexpression vector of the present invention into the host cell system. Insome embodiments, such methods are generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

In some embodiments, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant polypeptide. In some embodiments, effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Inone embodiment, an effective medium refers to any medium in which a cellis cultured to produce the recombinant polypeptide of the presentinvention. In some embodiments, a medium typically includes an aqueoussolution having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. In some embodiments, cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes and petri plates. In some embodiments,culturing is carried out at a temperature, pH and oxygen contentappropriate for a recombinant cell. In some embodiments, culturingconditions are within the expertise of one of ordinary skill in the art.

In some embodiments, depending on the vector and host system used forproduction, resultant polypeptides of the present invention eitherremain within the recombinant cell, secreted into the fermentationmedium, secreted into a space between two cellular membranes, such asthe periplasmic space in E. coli; or retained on the outer surface of acell or viral membrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant polypeptide is effected.

In one embodiment, the phrase “recovering the recombinant polypeptide”used herein refers to collecting the whole fermentation mediumcontaining the polypeptide and need not imply additional steps ofseparation or purification.

In one embodiment, polypeptides of the present invention are purifiedusing a variety of standard protein purification techniques, such as,but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the polypeptide of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the polypeptide can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the polypeptide and the cleavable moiety and the polypeptide canbe released from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, the polypeptide of the present invention is retrievedin “substantially pure” form.

In one embodiment, the phrase “substantially pure” refers to a puritythat allows for the effective use of the protein in the applicationsdescribed herein.

In one embodiment, the polypeptide of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In one embodiment, production of CTP-EPO-CTP polypeptides usingrecombinant DNA technology is illustrated in Example 1.

In some embodiments, the recombinant polypeptides are synthesized andpurified; their therapeutic efficacy can be assayed in vivo or in vitro.In one embodiment, the binding activities of the recombinant EPOpolypeptides of the present invention can be ascertained using variousassays as described in Examples 2-6 and 8-9. In one embodiment, in vitrobinding activity is ascertained by measuring the ability of thepolypeptide to stimulate proliferation of TF-1 cells. In one embodiment,in vivo activity is deduced by analyzing heamatocrit levels (FIGS. 3-5)and/or as a percentage of reticulocytes.

In one embodiment, the EPO polypeptides of the present invention can beused to treat a subject, with a variety of erythropoietin-associatedconditions. In some embodiments, a subject is a human subject.

In some embodiment, the phrase “erythropoietin-associated conditions”refers to any condition associated with below normal, abnormal, orinappropriate modulation of erythropoietin. In some embodiment, levelsof erythropoietin associated with such conditions are determined by anymeasure accepted and utilized by those of skill in the art. In someembodiment, erythropoietin-associated conditions typically includeanemic conditions.

In some embodiment, “anemic conditions” refers to any condition,disease, or disorder associated with anemia. In some embodiment, anemicconditions include, but are not limited to, aplastic anemia, autoimmunehemolytic anemia, bone marrow transplantation, Churg-Strauss syndrome,Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome, graft versushost disease, hematopoietic stem cell transplantation, hemolytic uremicsyndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpuraSchoenlein-Henoch, sideroblastic anemia, refractory anemia with excessof blasts, rheumatoid arthritis, Shwachman syndrome, sickle celldisease, thalassemia major, thalassemia minor, thrombocytopenic purpura,etc.

In one embodiment, production of GH modified by CTPs using recombinantDNA technology is preformed.

In some embodiments, the recombinant polypeptides are synthesized andpurified; their therapeutic efficacy can be assayed either in vivo or invitro. In one embodiment, the binding activities of the recombinant GHmodified by CTPs of the present invention can be ascertained usingvarious assays.

In one embodiment, the present invention comprises CTP-GH-CTPpolypeptides. In one embodiment, recombinant DNA technology methods areused for the production of CTP-GH-CTP polypeptides as illustrated inExample 7. In one embodiment, the therapeutic efficacy of the CTP-GH-CTPpolypeptides of the present invention is assayed either in vivo. In oneembodiment, the therapeutic efficacy of the CTP-GH-CTP polypeptides ofthe present invention is assayed either in vitro. In one embodiment, thebinding activities of the recombinant GH polypeptides of the presentinvention are measured using Nb2 (a prolactin-dependent rat lymphomacell line (ECACC Cell Bank)) or a FCD-P1 murine cell line, previouslytransfected with human growth hormone receptor. In one embodiment,binding of GH to these receptors induces cell proliferation which in oneembodiment is measured by the levels of MTT cellular stain as a functionof GH activity. In one embodiment, in vivo activity is deduced bymeasuring weight gain over time in treated growth hormone deficientanimals.

In one embodiment, the present invention provides a method of inducinggrowth or weight gain in a subject, comprising administering to thesubject a therapeutically effective amount of a polypeptide comprising agrowth hormone, one chorionic gonadotrophin carboxy terminal peptide(CTP) attached to an amino terminus of said growth hormone, and twochorionic gonadotrophin CTPs attached to a carboxy terminus of thegrowth hormone, thereby inducing growth or weight gain in a subject.

In another embodiment, the present invention provides a method ofinducing growth or weight gain in a non-human subject, comprising thestep of administering to said non-human subject a therapeuticallyeffective amount of an expression vector comprising a polynucleotideconsisting of a nucleic acid encoding a polypeptide, said polypeptideconsisting of a non-human growth hormone, one chorionic gonadotrophincarboxy terminal peptide (CTP) attached to the amino terminus of saidnon-human growth hormone, and two chorionic gonadotrophin CTPs attachedto the carboxy terminus of said non-human growth hormone, and whereinsaid polypeptide optionally consists of a signal peptide attached to theamino terminus of said one CTP, thereby inducing growth or weight gainin a non-human subject.

In another embodiment, the present invention provides a method ofinducing weight loss or decreasing body fat in a subject, comprisingadministering to said subject a therapeutically effective amount of apolypeptide comprising a growth hormone, one chorionic gonadotrophincarboxy terminal peptide (CTP) attached to the amino terminus of saidgrowth hormone, and two chorionic gonadotrophin CTPs attached to thecarboxy terminus of said growth hormone, thereby inducing weight loss ordecreasing body fat in said subject. In one embodiment, said subject isobese. In another embodiment, said subject is overweight.

In another embodiment, the present invention provides a method ofdecreasing body fat in a non-human subject, comprising administering tosaid subject a therapeutically effective amount of an expression vectorcomprising a polynucleotide, said polynucleotide consisting of anon-human growth hormone, one chorionic gonadotrophin carboxy terminalpeptide (CTP) attached to the amino terminus of said non-human growthhormone, and two chorionic gonadotrophin CTPs attached to the carboxyterminus of said non-human growth hormone, and wherein said polypeptideoptionally consists of a signal peptide attached to the amino terminusof said one CTP, thereby inducing growth or weight gain in a non-humansubject.

In another embodiment, the present invention provides a method ofdecreasing fat deposits in a subject. In another embodiment, the presentinvention provides a method of increasing muscle mass in a subject. Inanother embodiment, the present invention provides a method of promotingmuscle growth in a subject. In another embodiment, the present inventionprovides a method of increasing muscle to fat ratio. In anotherembodiment, the present invention provides a method of decreasing bodymass index (BMI) or Quetelet index.

In another embodiment, growth is measured by weight gain. In anotherembodiment, growth is measured by height gain. In another embodiment,growth is measured by weight gain. In another embodiment, growth ismeasured by muscle mass gain. In another embodiment, growth is measuredby weight gain. In another embodiment, growth is measured by bone massgain. In another embodiment, growth is measured by weight gain. Inanother embodiment, growth is measured by muscle mass gain. In anotherembodiment, the weight gain is due to bone and/or muscle mass gain. Inanother embodiment, growth is measured by any known measure known to oneof skill in the art.

In some embodiment, human growth hormone polypeptides of the presentinvention can be used to treat a subject, with conditions related togrowth and weight, such as a growth deficiency disorder, AIDS wasting,aging, impaired immune function of HIV-infected subjects, a catabolicillness, surgical recovery, a congestive cardiomyopathy, livertransplantation, liver regeneration after hepatectomy, chronic renalfailure, renal osteodystrophy, osteoporosis,achondroplasia/hypochondroplasia, skeletal dysplasia, a chronicinflammatory or nutritional disorder such as Crohn's disease, shortbowel syndrome, juvenile chronic arthritis, cystic fibrosis, maleinfertility, X-linked hypophosphatemic rickets, Down's syndrome, Spinabifida, Noonan Syndrome, obesity, impaired muscle strength andfibromyalgia. In some embodiments, interferon polypeptides of thepresent invention are used to treat a subject, with a variety ofconditions such as hairy cell leukemia (HCL), Kaposi's sarcoma (KS),chronic myelogenous leukemia (CML), chronic Hepatitis C (CHC),condylomata acuminata (CA), chronic Hepatitis B, malignant melanoma,follicular non-Hodgkin's lymphoma, multiple sclerosis, chronicgranulomatous disease, Mycobacterium avium complex (MAC), pulmonaryfibrosis and osteoporosis.

In some embodiments, Glucagon-like peptide-1 (GLP-1) polypeptides of thepresent invention are used to treat a subject with non-insulin dependentdiabetes, obesity, stroke, myocardial infarction, stroke, stress-inducedhyperglycemia, or irritable bowel syndrome.

In one embodiment, the polypeptides of the present invention can beprovided to the individual per se. In one embodiment, the polypeptidesof the present invention can be provided to the individual as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

In one embodiment, a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

In one embodiment, “active ingredient” refers to the polypeptidesequence of interest, which is accountable for the biological effect.

In some embodiments, any of the compositions of this invention willcomprise at least two CTP sequences bound to a protein of interest, inany form. In one embodiment, the present invention provides combinedpreparations. In one embodiment, “a combined preparation” definesespecially a “kit of parts” in the sense that the combination partnersas defined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partnersi.e., simultaneously, concurrently, separately or sequentially. In someembodiments, the parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partners, in some embodiments, can be administered in thecombined preparation. In one embodiment, the combined preparation can bevaried, e.g., in order to cope with the needs of a patient subpopulationto be treated or the needs of the single patient which different needscan be due to a particular disease, severity of a disease, age, sex, orbody weight as can be readily made by a person skilled in the art.

In one embodiment, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which can be used interchangeablyused refer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979).

In one embodiment, “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In one embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

In one embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body.

In another embodiment, polypeptides comprising GH modified by CTPs ofthe present invention are administered in a dose of 1-90 micrograms in0.1-5 ml solution. In another embodiment, polypeptides comprising GHmodified by CTPs are administered in a dose of 1-50 micrograms in 0.1-5ml solution. In another embodiment, polypeptides comprising GH modifiedby CTPs are administered in a dose of 1-25 micrograms in 0.1-5 mlsolution. In another embodiment, polypeptides comprising GH modified byCTPs are administered in a dose of 50-90 micrograms in 0.1-5 mlsolution. In another embodiment, polypeptides comprising GH modified byCTPs are administered in a dose of 10-50 micrograms in 0.1-5 mlsolution.

In another embodiment, polypeptides comprising GH modified by CTPs areadministered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (IM) injection, subcutaneous (SC) injection, orintravenous (IV) injection once a week. In another embodiment,polypeptides comprising GH modified by CTPs are administered in a doseof 1-90 micrograms in 0.1-5 ml solution by intramuscular (IM) injection,subcutaneous (SC) injection, or intravenous (IV) injection twice a week.In another embodiment, polypeptides comprising GH modified by CTPs areadministered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (IM) injection, subcutaneous (SC) injection, orintravenous (IV) injection three times a week. In another embodiment,polypeptides comprising GH modified by CTPs are administered in a doseof 1-90 micrograms in 0.1-5 ml solution by intramuscular (IM) injection,subcutaneous (SC) injection, or intravenous (IV) injection once everytwo weeks. In another embodiment, polypeptides comprising GH modified byCTPs are administered in a dose of 1-90 micrograms in 0.1-5 ml solutionby intramuscular (IM) injection, subcutaneous (SC) injection, orintravenous (IV) injection once every 17 days. In another embodiment,polypeptides comprising GH modified by CTPs are administered in a doseof 1-90 micrograms in 0.1-5 ml solution by intramuscular (IM) injection,subcutaneous (SC) injection, or intravenous (IV) injection once every 19days weeks.

Various embodiments of dosage ranges are contemplated by this invention.The dosage of the polypeptide of the present invention, in oneembodiment, is in the range of 0.05-80 mg/day. In another embodiment,the dosage is in the range of 0.05-50 mg/day. In another embodiment, thedosage is in the range of 0.1-20 mg/day. In another embodiment, thedosage is in the range of 0.1-10 mg/day. In another embodiment, thedosage is in the range of 0.1-5 mg/day. In another embodiment, thedosage is in the range of 0.5-5 mg/day. In another embodiment, thedosage is in the range of 0.5-50 mg/day. In another embodiment, thedosage is in the range of 5-80 mg/day. In another embodiment, the dosageis in the range of 35-65 mg/day. In another embodiment, the dosage is inthe range of 35-65 mg/day. In another embodiment, the dosage is in therange of 20-60 mg/day. In another embodiment, the dosage is in the rangeof 40-60 mg/day. In another embodiment, the dosage is in a range of45-60 mg/day. In another embodiment, the dosage is in the range of 40-60mg/day. In another embodiment, the dosage is in a range of 60-120mg/day. In another embodiment, the dosage is in the range of 120-240mg/day. In another embodiment, the dosage is in the range of 40-60mg/day. In another embodiment, the dosage is in a range of 240-400mg/day. In another embodiment, the dosage is in a range of 45-60 mg/day.In another embodiment, the dosage is in the range of 15-25 mg/day. Inanother embodiment, the dosage is in the range of 5-10 mg/day. Inanother embodiment, the dosage is in the range of 55-65 mg/day.

In one embodiment, the dosage is 20 mg/day. In another embodiment, thedosage is 30 mg/day. In another embodiment, the dosage is 40 mg/day. Inanother embodiment, the dosage is 50 mg/day. In another embodiment, thedosage is 60 mg/day. In another embodiment, the dosage is 70 mg/day. Inanother embodiment, the dosage is 80 mg/day. In another embodiment, thedosage is 90 mg/day. In another embodiment, the dosage is 100 mg/day.

The dosage of the GH modified by CTPs of the present invention, in oneembodiment, is in the range of 0.005-100 mg/week. In another embodiment,the dosage is in the range of 0.005-5 mg/week. In another embodiment,the dosage is in the range of 0.01-50 mg/week. In another embodiment,the dosage is in the range of 0.1-20 mg/week. In another embodiment, thedosage is in the range of 0.1-10 mg/week. In another embodiment, thedosage is in the range of 0.01-5 mg/week. In another embodiment, thedosage is in the range of 0.001-0.01 mg/week. In another embodiment, thedosage is in the range of 0.001-0.1 mg/week. In another embodiment, thedosage is in the range of 0.1-5 mg/week. In another embodiment, thedosage is in the range of 0.5-50 mg/week. In another embodiment, thedosage is in the range of 0.2-15 mg/week. In another embodiment, thedosage is in the range of 0.8-65 mg/week. In another embodiment, thedosage is in the range of 1-50 mg/week. In another embodiment, thedosage is in the range of 5-10 mg/week. In another embodiment, thedosage is in the range of 8-15 mg/week. In another embodiment, thedosage is in a range of 10-20 mg/week. In another embodiment, the dosageis in the range of 20-40 mg/week. In another embodiment, the dosage isin a range of 60-120 mg/week. In another embodiment, the dosage is inthe range of 12-40 mg/week. In another embodiment, the dosage is in therange of 40-60 mg/week. In another embodiment, the dosage is in a rangeof 50-100 mg/week. In another embodiment, the dosage is in a range of1-60 mg/week. In another embodiment, the dosage is in the range of 15-25mg/week. In another embodiment, the dosage is in the range of 5-10mg/week. In another embodiment, the dosage is in the range of 55-65mg/week. In another embodiment, the dosage is in the range of 1-5mg/week.

In another embodiment, the GH dosage given to a subject is 50% of thestandard dosage given to a reference subject from the same population ofsubjects (e.g. children, elderly, men, women, GH deficient, specificnationality, etc). In another embodiment, the dosage is 30% of thedosage given to a subject from a specific population of subjects. Inanother embodiment, the dosage is 45% of the dosage given to a subjectfrom a specific population of subjects. In another embodiment, thedosage is 100% of the dosage given to a subject from a specificpopulation of subjects.

In another embodiment, the dosage is 1-5 mg/week. In another embodiment,the dosage is 2 mg/week. In another embodiment, the dosage is 4 mg/week.In another embodiment, the dosage is 1.2 mg/week. In another embodiment,the dosage is 1.8 mg/week. In another embodiment, the dosage isapproximately the dosages described herein.

In another embodiment, the dosage is 1-5 mg/administration. In anotherembodiment, the dosage is 2 mg/administration. In another embodiment,the dosage is 4 mg/administration. In another embodiment, the dosage is1.2 mg/administration. In another embodiment, the dosage is 1.8mg/administration. In one embodiment, the composition is administeredonce a week. In another embodiment, the composition is administered oncebiweekly. In another embodiment, the composition is administeredmonthly. In another embodiment, the composition is administered daily.

In another embodiment, GH modified by CTPs is formulated in anintranasal dosage form. In another embodiment, GH modified by CTPs isformulated in an injectable dosage form. In another embodiment, GHmodified by CTPs is administered to a subject in a dose ranging from0.0001 mg to 0.6 mg. In another embodiment, GH modified by CTPs isadministered to a subject in a dose ranging from 0.001 mg to 0.005 mg.In another embodiment, GH modified by CTPs is administered to a subjectin a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, GHmodified by CTPs is administered to a subject in a dose ranging from0.01 mg to 0.3 mg. In another embodiment, a GH modified by CTPs isadministered to a subject in a dose in a dose ranging from 0.2 mg to 0.6mg.

In another embodiment, GH modified by CTPs is administered to a subjectin a dose ranging from 1-100 micrograms. In another embodiment, a GHmodified by CTPs is administered to a subject in a dose ranging from10-80 micrograms. In another embodiment, a GH modified by CTPs isadministered to a subject in a dose ranging from 20-60 micrograms. Inanother embodiment, a GH modified by CTPs is administered to a subjectin a dose ranging from 10-50 micrograms. In another embodiment, a GHmodified by CTPs is administered to a subject in a dose ranging from40-80 micrograms. In another embodiment, a GH modified by CTPs isadministered to a subject in a dose ranging from 10-30 micrograms. Inanother embodiment, a GH modified by CTPs is administered to a subjectin a dose ranging from 30-60 micrograms.

In another embodiment, GH modified by CTPs is administered to a subjectin a dose ranging from 0.2 mg to 2 mg. In another embodiment, a GHmodified by CTPs is administered to a subject in a dose ranging from 2mg to 6 mg. In another embodiment, a GH modified by CTPs is administeredto a subject in a dose ranging from 4 mg to 10 mg. In anotherembodiment, a GH modified by CTPs is administered to a subject in a doseranging from 5 mg and 15 mg.

In another embodiment, a GH modified by CTPs is injected into the muscle(intramuscular injection). In another embodiment, a GH modified by CTPsis injected below the skin (subcutaneous injection). In anotherembodiment, a GH modified by CTPs is injected into the muscle. Inanother embodiment, a GH modified by CTPs is injected below the skin.

In another embodiment, the methods of the invention include increasingthe compliance in the use of GH therapy, comprising providing to asubject in need thereof, a GH modified by CTPs, thereby increasingcompliance in the use of growth hormone therapy.

In another embodiment, protein drugs of molecular weight lower than50,000 daltons, such as GH modified by CTPs of the present invention arein general short-lived species in vivo, having short circulatoryhalf-lives of several hours. In another embodiment, the subcutaneousroute of administration in general provides slower release into thecirculation. In another embodiment, the CTP modified polypeptide of theinvention prolongs the half-live of protein drugs of molecular weightlower than 50,000 daltons, such as GH. In another embodiment, the CTPmodified polypeptide of the invention enable interferons to exert theirbeneficial effects for a longer period of time.

In another embodiment, the immunogenicity of a CTP modified polypeptidecomprising a GH modified by CTPs is equal to an isolated GH. In anotherembodiment, the immunogenicity of a CTP modified polypeptide comprisinga GH modified by CTPs is comparable to an isolated GH. In anotherembodiment, modifying a GH as described herein with CTP peptides reducesthe immunogenicity of the GH. In another embodiment, the CTP modifiedpolypeptide comprising a GH is as active as an isolated GH protein. Inanother embodiment, the CTP modified polypeptide comprising a GH is moreactive than an isolated GH. In another embodiment, the CTP modifiedpolypeptide comprising a GH maximizes the growth hormone's protectiveability against degradation while minimizing reductions in bioactivity.

In another embodiment, the methods of the invention include increasingthe compliance of subjects afflicted with chronic illnesses that are inneed of a GH therapy. In another embodiment, the methods of theinvention enable reduction in the dosing frequency of a GH by modifyingthe GH with CTPs as described hereinabove. In another embodiment, theterm compliance comprises adherence. In another embodiment, the methodsof the invention include increasing the compliance of patients in needof a GH therapy by reducing the frequency of administration of the GH.In another embodiment, reduction in the frequency of administration ofthe GH is achieved due to the CTP modifications which render theCTP-modified GH more stable. In another embodiment, reduction in thefrequency of administration of the GH is achieved as a result ofincreasing T1/2 of the growth hormone. In another embodiment, reductionin the frequency of administration of the GH is achieved as a result ofincreasing the clearance time of the GH. In another embodiment,reduction in the frequency of administration of the growth hormone isachieved as a result of increasing the AUC measure of the growthhormone.

In another embodiment, the present invention provides a method ofdecreasing body fat in a non-human subject, comprising administering tosaid subject a therapeutically effective amount of an expression vectorcomprising a polynucleotide, said polynucleotide consisting of anon-human growth hormone, one chorionic gonadotrophin carboxy terminalpeptide (CTP) attached to the amino terminus of said non-human growthhormone, and two chorionic gonadotrophin CTPs attached to the carboxyterminus of said non-human growth hormone, and wherein said polypeptideoptionally consists of a signal peptide attached to the amino terminusof said one CTP, thereby inducing growth or weight gain in a non-humansubject.

In another embodiment, the present invention provides a method ofincreasing insulin-like growth factor (IGF-1) levels in a human subject,comprising administering to said subject a therapeutically effectiveamount of a polypeptide comprising a growth hormone, one chorionicgonadotrophin carboxy terminal peptide (CTP) attached to the aminoterminus of said growth hormone, and two chorionic gonadotrophin CTPsattached to the carboxy terminus of said growth hormone, therebyincreasing IGF-1 levels in said subject.

In another embodiment, the present invention provides a method ofincreasing insulin-like growth factor (IGF-1) levels in a non-humansubject, comprising administering to said subject a therapeuticallyeffective amount of an expression vector comprising a polynucleotide,said polynucleotide consisting of a non-human growth hormone, onechorionic gonadotrophin carboxy terminal peptide (CTP) attached to theamino terminus of said non-human growth hormone, and two chorionicgonadotrophin CTPs attached to the carboxy terminus of said non-humangrowth hormone, and wherein said polypeptide optionally consists of asignal peptide attached to the amino terminus of said one CTP, therebyinducing growth or weight gain in a non-human subject.

In one embodiment, increasing IGF-1 levels in a human subject may beeffective in treating, preventing or suppressing type 1 diabetes, type 2diabetes, amyotrophic lateral sclerosis (ALS aka “Lou Gehrig'sDisease”), severe burn injury and myotonic muscular dystrophy (MMD).

In another embodiment, a GH modified by CTPs is administered to asubject once a day. In another embodiment, a polypeptide comprising a GHmodified by CTPs is administered to a subject once every two days. Inanother embodiment, a GH modified by CTPs is administered to a subjectonce every three days. In another embodiment, a GH modified by CTPs isadministered to a subject once every four days. In another embodiment, aGH modified by CTPs is administered to a subject once every five days.In another embodiment, a GH modified by CTPs is administered to asubject once every six days. In another embodiment, a GH modified byCTPs is administered to a subject once every week. In anotherembodiment, a GH modified by CTPs is administered to a subject onceevery 7-14 days. In another embodiment, a GH modified by CTPs isadministered to a subject once every 10-20 days. In another embodiment,a GH modified by CTPs is administered to a subject once every 5-15 days.In another embodiment, a GH modified by CTPs is administered to asubject once every 15-30 days.

In another embodiment, the dosage is in a range of 50-500 mg/day. Inanother embodiment, the dosage is in a range of 50-150 mg/day. Inanother embodiment, the dosage is in a range of 100-200 mg/day. Inanother embodiment, the dosage is in a range of 150-250 mg/day. Inanother embodiment, the dosage is in a range of 200-300 mg/day. Inanother embodiment, the dosage is in a range of 250-400 mg/day. Inanother embodiment, the dosage is in a range of 300-500 mg/day. Inanother embodiment, the dosage is in a range of 350-500 mg/day.

In one embodiment, the dosage is 20 mg/day. In one embodiment, thedosage is 30 mg/day. In one embodiment, the dosage is 40 mg/day. In oneembodiment, the dosage is 50 mg/day. In one embodiment, the dosage is0.01 mg/day. In another embodiment, the dosage is 0.1 mg/day. In anotherembodiment, the dosage is 1 mg/day. In another embodiment, the dosage is0.530 mg/day. In another embodiment, the dosage is 0.05 mg/day. Inanother embodiment, the dosage is 50 mg/day. In another embodiment, thedosage is 10 mg/day. In another embodiment, the dosage is 20-70 mg/day.In another embodiment, the dosage is 5 mg/day.

In another embodiment, the dosage is 1-90 mg/day. In another embodiment,the dosage is 1-90 mg/2 days. In another embodiment, the dosage is 1-90mg/3 days. In another embodiment, the dosage is 1-90 mg/4 days. Inanother embodiment, the dosage is 1-90 mg/5 days. In another embodiment,the dosage is 1-90 mg/6 days. In another embodiment, the dosage is 1-90mg/week. In another embodiment, the dosage is 1-90 mg/9 days. In anotherembodiment, the dosage is 1-90 mg/11 days. In another embodiment, thedosage is 1-90 mg/14 days.

In another embodiment, the growth hormone dosage is 10-50 mg/day. Inanother embodiment, the dosage is 10-50 mg/2 days. In anotherembodiment, the dosage is 10-50 mg/3 days. In another embodiment, thedosage is 10-50 mg/4 days. In another embodiment, the dosage is 10-50micrograms mg/5 days. In another embodiment, the dosage is 10-50 mg/6days. In another embodiment, the dosage is 10-50 mg/week. In anotherembodiment, the dosage is 10-50 mg/9 days. In another embodiment, thedosage is 10-50 mg/11 days. In another embodiment, the dosage is 10-50mg/14 days.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of the desired compound, or compounds, each of which isin one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or inanother embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. Thepharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmellose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the pharmaceutical activeingredient as known to one skilled in the art.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like. In some embodiments,pharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. In some embodiments, liquid oralcompositions comprise from about 0.012% to about 0.933% of the desiredcompound or compounds, or in another embodiment, from about 0.033% toabout 0.7%.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofthe compounds of the present invention and optionally, other compounds,intended for topical intranasal administration. In some embodiments, hcompositions comprise from about 0.01% to about 10.0% w/v of a subjectcompound, more preferably from about 0.1% to about 2.0, which is usedfor systemic delivery of the compounds by the intranasal route.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. In some embodiments, liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intra-arterially, and are thus formulatedin a form suitable for intra-arterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces, and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the compounds of the present invention are combined withan additional appropriate therapeutic agent or agents, prepared andapplied as solutions, suspensions, or emulsions in a physiologicallyacceptable diluent with or without a pharmaceutical carrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientsinto preparations which, can be used pharmaceutically. In oneembodiment, formulation is dependent upon the route of administrationchosen.

In one embodiment, injectables, of the invention are formulated inaqueous solutions. In one embodiment, injectables, of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in oily or aqueous vehicles, andcontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In some embodiments, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of the active ingredients,in some embodiments, are prepared as appropriate oily or water basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contain suitable stabilizers or agents which increase thesolubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990).

In some embodiments, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use. Compositions are formulated, in someembodiments, for atomization and inhalation administration. In anotherembodiment, compositions are contained in a container with attachedatomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In some embodiments, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. In some embodiments, a therapeutically effective amount meansan amount of active ingredients effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

The compositions also comprise preservatives, such as benzalkoniumchloride and thimerosal and the like; chelating agents, such as edetatesodium and others; buffers such as phosphate, citrate and acetate;tonicity agents such as sodium chloride, potassium chloride, glycerin,mannitol and others; antioxidants such as ascorbic acid, acetylcystine,sodium metabisulfote and others; aromatic agents; viscosity adjustors,such as polymers, including cellulose and derivatives thereof; andpolyvinyl alcohol and acid and bases to adjust the pH of these aqueouscompositions as needed. The compositions also comprise local anestheticsor other actives. The compositions can be used as sprays, mists, drops,and the like.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of apharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the pharmaceutically-acceptable carrier is sterile,physiological saline, with a blood-compatible suspending agent, the pHof which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In some embodiments, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compounds solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In some embodiments, preparation of an effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the activeingredients described herein can be determined by standardpharmaceutical procedures in vitro, in cell cultures or experimentalanimals. In one embodiment, the data obtained from these in vitro andcell culture assays and animal studies can be used in formulating arange of dosage for use in human. In one embodiment, the dosages varydepending upon the dosage form employed and the route of administrationutilized. In one embodiment, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. [See e.g., Fingl, et al., (1975) “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In another embodiment, a GH modified by CTPs is administered viasystemic administration. In another embodiment, a growth hormone asdescribed herein is administered by intravenous, intramuscular orsubcutaneous injection. In another embodiment, a GH modified by CTPs islyophilized (i.e., freeze-dried) preparation in combination with complexorganic excipients and stabilizers such as nonionic surface activeagents (i.e., surfactants), various sugars, organic polyols and/or humanserum albumin. In another embodiment, a pharmaceutical compositioncomprises a lyophilized GH modified by CTPs as described in sterilewater for injection. In another embodiment, a pharmaceutical compositioncomprises a lyophilized growth hormone as described in sterile PBS forinjection. In another embodiment, a pharmaceutical composition comprisesa lyophilized growth hormone as described in sterile 0.9% NaCl forinjection.

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein and complex carriers such as humanserum albumin, polyols, sugars, and anionic surface active stabilizingagents. See, for example, WO 89/10756 (Hara et al.—containing polyol andp-hydroxybenzoate). In another embodiment, the pharmaceuticalcomposition comprises a growth hormone as described herein andlactobionic acid and an acetate/glycine buffer. In another embodiment,the pharmaceutical composition comprising a GH modified by CTPs asdescribed herein and amino acids, such as arginine or glutamate thatincrease the solubility of interferon compositions in water. In anotherembodiment, the pharmaceutical composition comprises a lyophilized GHmodified by CTPs as described herein and glycine or human serum albumin(HSA), a buffer (e g. acetate) and an isotonic agent (e.g NaCl). Inanother embodiment, the pharmaceutical composition comprises alyophilized GH modified by CTPs as described herein and phosphatebuffer, glycine and HSA.

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein is stabilized when placed inbuffered solutions having a pH between about 4 and 7.2. In anotherembodiment, the pharmaceutical composition comprising a GH modified byCTPs as described herein is stabilized with an amino acid as astabilizing agent and in some cases a salt (if the amino acid does notcontain a charged side chain).

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein is a liquid composition comprisinga stabilizing agent at between about 0.3% and 5% by weight which is anamino acid.

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein provides dosing accuracy andproduct safety. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein provides abiologically active, stable liquid formulation for use in injectableapplications. In another embodiment, the pharmaceutical compositioncomprises a non-lyophilized GH modified by CTPs as described herein.

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein provides a liquid formulationpermitting storage for a long period of time in a liquid statefacilitating storage and shipping prior to administration.

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein comprises solid lipids as matrixmaterial. In another embodiment, the injectable pharmaceuticalcomposition comprising a GH modified by CTPs as described hereincomprises solid lipids as matrix material. In another embodiment, theproduction of lipid microparticles by spray congealing was described bySpeiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipidnanopellets for peroral administration (Speiser EP 0167825 (1990)). Inanother embodiment, lipids, which are used, are well tolerated by thebody (e. g. glycerides composed of fatty acids which are present in theemulsions for parenteral nutrition).

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein is in the form of liposomes (J. E.Diederichs and al., Pharm./nd. 56 (1994) 267-275).

In another embodiment, the pharmaceutical composition comprising a GHmodified by CTPs as described herein comprises polymeric microparticles.In another embodiment, the injectable pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprises polymericmicroparticles. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprisesnanoparticles. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprisesliposomes. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprises lipidemulsion. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprisesmicrospheres. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprises lipidnanoparticles. In another embodiment, the pharmaceutical compositioncomprising a GH modified by CTPs as described herein comprises lipidnanoparticles comprising amphiphilic lipids. In another embodiment, thepharmaceutical composition comprising a GH modified by CTPs as describedherein comprises lipid nanoparticles comprising a drug, a lipid matrixand a surfactant. In another embodiment, the lipid matrix has amonoglyceride content which is at least 50% w/w.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the active ingredient.In one embodiment, the pack, for example, comprise metal or plasticfoil, such as a blister pack. In one embodiment, the pack or dispenserdevice is accompanied by instructions for administration. In oneembodiment, the pack or dispenser is accommodated by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor human or veterinary administration. Such notice, in one embodiment,is labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert.

In one embodiment, it will be appreciated that the GH modified by CTPsof the present invention can be provided to the individual withadditional active agents to achieve an improved therapeutic effect ascompared to treatment with each agent by itself. In another embodiment,measures (e.g., dosing and selection of the complementary agent) aretaken to minimize adverse side effects which are associated withcombination therapies.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Example 1 Generation of EPO Constructs

Materials and Methods:

Construction of expression vector pCI-dhfr: pCI-neo mammalian expressionvector was purchased from Promega (Catalog No. E1841). The vectorcontains a CMV IE enhancer/promoter and neomycin phosphotransferasegene. The pSV2-dhfr clone was purchased from ATCC (Catalog No. 37146).The plasmid contains the murine dhfr gene. The construction of pCI-dhfrvector was performed as follows:

-   -   a. The pSV2-dhfr plasmid was digested with restriction enzyme        BglII (3′ end of the dhfr gene). DNA polymerase I, Large        (Klenow) Fragment was used to fill-in the 5′ overhangs to form        blunt ends. The DNA was then digested with restriction enzyme        AvrII (5′ end of the dhfr gene). The dhfr gene (AvrII-blunt end)        fragment was isolated.    -   b. The pCI-neo vector was digested with restriction enzyme BstXI        (3′ end of the neo gene). DNA polymerase I, Large (Klenow)        Fragment was used to remove the 3′ overhangs to form blunt ends.        The DNA was then digested with restriction enzyme AvrII (5′ end        of the neo gene). The expression vector (AvrII-blunt end) was        isolated.    -   c. The dhfr gene was ligated into pCI vector to form an        expression vector containing the dhfr gene (pCI-dhfr).

Construction of hEPO-CTP variants: A cassette gene containing theC-Terminal peptide (CTP) of the beta subunit of hCG was fused to thecoding sequence of human EPO (NP_000790.2) at different locations. FourEPO-CTP variants were constructed as illustrated in FIGS. 1A-D. TheproEPO signal peptide was used for the construction of the secretedEPO-CTP variants. XbaI-NotI fragments containing Epo sequences wereligated into the pCI-dhfr expression vector of the present invention.

Table 2 hereinbelow summarizes the primer sequences used forconstructing the CTP-containing polypeptides of the present invention.

TABLE 2 Restric- tion site SEQ (under- Primer ID lined in  number NOsequence sequence)  1  7 5′ AATCTAGAGGTCATCATGGGGGT XbaI GC 3′  2  8 5′ATTGCGGCCGCGGATCCAGAAGA NotI CCTTTATTG 3′ 17^(R) 9 5′TAAATATTGGGGTGTCCGAGGGC SspI CC 3′ 10  10 5′ CCAATATTACCACAAGCCCCACCSspI ACGCCTCAT 3′ 11^(R) 11 5′ TGCGGCCGCGGATCCTTATCTGT NotICCCCTGTCCTGC 3′ 15  12 5′ GCCCTGCTGTCGGAAGC 3′  2^(R) 13 5′ATTGCGGCCGCGGATCCAGAAGA NotI CCTTTATTG 23^(R) 14 5′CTTTGAGGAAGAGGAGCCCAGGA CTGGGAGGC 3′ 24  15 5′ CCTGGGCTCCTCTTCCTCAAAGGC 3′ 38^(R) 50 5′ GCTTCCGACAGCAGGGC 3′

EPO-1 701-1-p17-6 (Epo-1—SEQ ID NO: 1): The XbaI-NotI 702 bp fragmentwas constructed by PCR using the above primers (SEQ ID NOs: 7-16). Thenthe XbaI-NotI PCR fragment containing Epo-ctp sequence was ligated intopCI-dhfr expression vector.

EPO-2 701-2-p24-2 (Epo-2—SEQ ID NO: 2): The XbaI/ApaI fragment (hGH-ctp)of pCI-dhfr-401-2-p21-2 (hGH-ctpx2) was replaced by the XbaI/ApaIfragment (EPO-ctp) of 701-1-p17-6 to create an Epo-ctpx2.

EPO-4-701-4-p42-1 (Epo-4—SEQ ID NO: 4): Firstly, a fragment frompCI-dhfr-EPO-ctp (701-1-p17-6) was constructed by PCR using primers 1and 17 followed by XbaI/SspI digestion. This resulted in a fragmentcontaining EPO and partial 5′ CTP.

Secondly, a new fragment was constructed by overlapping PCR, onpGT123-hEpo as a template, using primer 10 and primer 11. SspI/NotIdigestion resulted in a fragment containing 3′ partial CTP and Epo.

The two fragments were ligated into pCI-dhfr to construct thep701-4-p42-1 clone.

EPO-3-p56-6 (Epo-3 SEQ ID NO: 3): Primers were purchased fromSigma-Genosys. PCR reaction was performed using primer 15 (SEQ ID NO:12) and primer 2^(R) (SEQ ID NO: 13) and plasmid DNA of pCI-dhfr-EPO-ctpx2 (701-2-p24-2) as a template. As a result of the PCR amplification, a486 bp product was formed and ligated into a TA cloning vector(Invitrogen, catalog K2000-01). Stu I-NotI fragment containing *Epo-ctpx2 sequence was isolated (209 bp).

Three sequential PCR reactions were performed. The first reaction wasconducted with primer 1 (SEQ ID NO: 7) and primer 23^(R) (SEQ ID NO: 14)and plasmid DNA of pGT123-epo-ctp as a template; as a result of the PCRamplification, an 80 bp product was formed (signal peptide).

The second reaction was conducted with primer 24 (SEQ ID NO: 15) andprimer 11^(R) (SEQ ID NO: 11) and plasmid DNA of 701-4-p42-1 as atemplate; as a result of the PCR amplification, a 610 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 11^(R)(SEQ ID NO: 11) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 700 bpproduct was formed and the XbaI-StuI fragment was isolated.

The two fragments (XbaI-StuI and StuI-NotI) were inserted into theeukaryotic expression vector pCI-dhfr (triple ligation) to yield the701-3-p56-6 clone.

EPO-5-p91-4 (Epo-5 SEQ ID NO: 5—(ctp-Epo): Primers were ordered fromSigma-Genosys. A PCR reaction was performed using primer 1 (SEQ ID NO:7) and primer 11^(R) (SEQ ID NO: 11) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp x2 (701-3-p56-6) as a template; as a result of thePCR amplification, a 670 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). XbaI-NotI fragmentcontaining ctp-Epo sequence was ligated into our eukaryotic expressionvector pCI-dhfr to yield the 701-5-p91-4 clone.

EPO-6-p90-1 (Epo-6 SEQ ID NO: 6—(ctp-Epo-ctp): Three PCR reactions wereperformed. The first reaction was conducted with primer 1 (SEQ ID NO: 7)and primer 38^(R) (SEQ ID NO: 50) and plasmid DNA of 701-3-p56-6 as atemplate; as a result of the PCR amplification, a 400 bp product wasformed.

The second reaction was conducted with primer 15 (SEQ ID NO: 12) andprimer 2^(R) (SEQ ID NO: 13) and plasmid DNA of 701-1-p17-6 as atemplate; as a result of the PCR amplification, a 390 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 2^(R)(SEQ ID NO: 13) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 787 bpproduct was formed and ligated into TA cloning vector (Invitrogen,catalog K2000-01). The XbaI-NotI fragment containing ctp-Epo-ctpsequence was ligated into the eukaryotic expression vector pCI-dhfr toyield the 701-6-p90-1 clone.

Example 2 Expression and Isolation of EPO-CTP Polypeptides

Materials and Methods

DNA transfection and clone selection: DG44 cells were transfected withpCI-DHFR expression vectors containing EPO-CTP variants using FuGENE6Reagent (FuGENE Transfection Reagent—Roche Cat. 11 815 091 001). 48 hrfollowing transfection, cells were diluted and seeded at 50-200 cellsper well in a selective medium (CD DG44 Medium w/o HT (Gibco: Scotlandpart: #07990111A) Sku num.:ME060027 supplemented with 8 mM L-GlutamineBiological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68solution (Gibco: Cat: 240040-032). Selected clones were screened forhighest protein production using commercial ELISA. Three to fiveproducing clones per each variant were frozen for a backup cell bank. Aselected clone for each variant was adapted to growth in larger scalecultures up to 1 L flasks on an orbital shaker platform. Supernatantswere collected and analyzed by ELISA, SDS-PAGE and Western blot.Following the withdrawal of aliquots, the protein-containingsupernatants were kept frozen until further use.

Cell culture: DG44 cells were maintained in DG44 medium with HT(cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (BiologicalIndustries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution(Gibco: Cat: 240040-032), at 37° C. in humidified 8% CO₂ incubator.Transfected clones were maintained in DG44 basal medium without HTsupplement, hypoxanthine and thymidine, with pluronic acid andL-glutamine.

Sample preparation: Supernatants were collected, filtrated and analyzedby ELISA to determine protein concentration. SDS-PAGE and Western blotwere used to determine purity and identity. Following ELISA, sampleconcentrations were defined and the solution was dialyzed against PBS.Following the withdrawal of aliquots, the protein-contained supernatantswere kept frozen at −20° C. until further use.

Western blotting: Samples were electrophoresed on nondenaturing 15%SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins weretransferred to a 0.2 μm pore size nitrocellulose membrane (Sigma, SaintLouis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresiscell (Biorad Laboratories, Richmond, Calif.). The nitrocellulosemembrane was incubated in 5% non-fat dry milk for 2 h at roomtemperature. The membrane was incubated with EPO anti-serum (1:1000titer) overnight at 4° C. followed by three consecutive washes in PBScontaining 0.1% Tween (10 min/wash). The membrane was incubated withsecondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed,San Francisco, Calif.) for 2 h at room temperature, followed by threewashes. Finally, the nitrocellulose paper was reacted with enhancedchemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min,dried with a Whatman sheet, and exposed to X-ray film.

Results

Table 3 hereinbelow shows the concentrations of the various CTP-modifiedEPO forms obtained from 5 selected clones and their preparation forfurther testing.

TABLE 3 Post dilution in Mock sup. Stock according to Post ultra- TiterEpo3 titer filtration #Version # Clone IU/ml¹ IU/ml² IU/ml³ Epo0 17 3093102 335 SEQ ID NO: 16 Epo1 47 1049 104 291 SEQ ID NO: 1 Epo2 67 2160 110303 SEQ ID NO: 2 Epo3 85 105 119 392 SEQ ID NO: 3 Epo4 112 6100 ND 342SEQ ID NO: 4

-   1. EPO variants stock concentration were determined by ELISA    (Quantikine IVD Epo ELISA, DEPOO, R&D Systems)-   2. Samples EPO-0, 1, 2 and 4 were diluted to 105 IU/ml in mock sup    (Adjusted to Epo3 titer). Epo0=wild type EPO expressed in the same    system as the CTP-modified EPOs.-   3. All samples were concentrated and dialyzed by ultrafiltration    against PBS to a final concentration of 180 IU/ml.

All proteins were detected by Western blot as illustrated in FIG. 2.

Example 3 Biological Activity of the EPO-CTP Polypeptides of the PresentInvention

The TF-1 bioactivity test represents the ability of the EPO-CTP variantsto bind its receptor and then stimulate activity which results in cellproliferation. Therefore, this test was used as a first step inevaluating the biological potency of the EPO-CTP polypeptides of thepresent invention.

Materials and Methods

Cell Proliferation Analysis: Proliferation assay was performed with thecell line TF-1, measuring levels of MTT cellular stain as a function ofEPO activity (Kitamura et al., Kitamura, T. et al. (1989) Establishmentand characterization of a unique human cell line that proliferates;Hammerling U. et al. In vitro bioassay for human erythropoietin based onproliferative stimulation of an erythroid cell line and analysis ofcarbohydrate-dependent microheterogeneity. Journal of Pharm. Biomed.Analysis 14(11): 1455-1469 (1996). Exponentially growing TF-1 cells werewashed twice, plated at about 10⁴ cells/well in microtiter plates, andincubated in basal medium with a titrated dilution series of EPO(Recormon®), EPO standard (NIBSC standard), rhEPO (MOD-7010), MOD-701variants (EPO-1, EPO-2, EPO-3 and EPO-4) for 48 hours. 4 hours prior toassaying for cell proliferation, MTT reagent was added to the wells, andabsorbance was measured by ELISA reader. A calculated proteinconcentration value for each variant protein was obtained from Eprex's(Epoetin (EPO)-man-made form of the human hormone) dose-responsestandard curve.

Results

The in vitro biological activity of EPO polypeptides was determined withan Epo-dependent cell line, human erythroleukemia TF-1 (DSMZ Cell Bank)[Dong et al., Biochemical and Biophysical Research Communications,Volume 339, Issue 1, 6 Jan. 2006, Pages 380-385]. The MTT assay wasperformed [Hammerling U. et al. In vitro bioassay for humanerythropoietin based on proliferative stimulation of an erythroid cellline and analysis of carbohydrate-dependent microheterogeneity. Journalof Pharm. Biomed. Analysis 14(11): 1455-1469 (1996);], and thelaboratory standard of EPO used to generate the standard curve wascalibrated against the International Standard (Epo ampoule code 87/684of NIBSC).

The results are summarized in Table 4 hereinbelow. The results indicatethat the highest potency was achieved by using EPO 3 and EPO 0 in both 2and 0.5 IU/ml concentrations.

TABLE 4 TF-1 Bioactivity IU/ml EPO 0 EPO 1 EPO 2 EPO 3 EPO 4 Eprex STDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID IU/ml NO: 16 NO: 1 NO: 2 NO: 3 NO: 4Recormon ® EPO st 2 4.93 2.32 2.13 6.91 3.55 3.44 7.40 0.5 1.60 0.760.53 1.34 0.84 0.87 1.53Conclusion

As summarized in Table 4 hereinabove, different levels of potency wereexerted by EPO-CTP polypeptides, indicating differences in receptorbinding. EPO-CTP polypeptides differ by the number of CTP cassettes andthe location to which they are fused. EPO-1 and EPO-2 contain 1 CTPsequence or 2 CTP sequences at the C-terminal of EPO, while EPO-3contains 1 CTP at N-terminal and 2 CTP sequences at C-terminal EPO-4 isa dimer of two EPO molecules linked by CTP sequence. EPO-3 demonstratedpotency level quite similar to WT-EPO, while EPO-1 and EPO-4 were about50% less potent than WT-EPO, and EPO-2 potency was even less than 50%.

Example 4 Evaluation of the EPO-CTP Polypeptides of the PresentInvention in a Mouse Model

The following experiment was performed in order to compare thebio-activity of the EPO-CTP polypeptides of the present invention andcommercial EPO

Materials and Methods

Animals:

-   Species/Strain: ICR or CD-1 Mice of either sex about 20-25 g-   Group Size: n=7-   No. Groups: 9-   Total No. Animals: n=63

Experimental design of the study: The experiment was set up assummarized in Table 5 hereinbelow.

TABLE 5 Group No. Mice TREATMENT Dosing No. per Group Compound DoseLevel Regimen 1 n = 7 Vehicle (Control) 0 1x weekly 2 MOCK 3 MOD-7010 15μg/kg 4 MOD-7011 5 MOD-7012 6 MOD-7013 7 MOD-7014 8 Commercial 15 μg/kg9 rhEPO 5 μg/kg 3 x weekly

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 22 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countwas carried out in all test animals at day 2 and 14 hrs following the1st respective vehicle or treatment injection. HCT was determined in allanimals once prior to initial treatment (“0” Baseline control) and at 24hrs after the 1st respective vehicle or treatment injection, andthereafter twice weekly until study termination (Day-22).

Results

The hematocrit results which are illustrated in FIGS. 3-5 show that EPO3 has the highest hematocrit percentage change from baseline compared toEPO 1, EPO 2, Recormon® 1, Recormon® 3, rhEPO, and vehicle. The resultsdemonstrating the percentage of reticulocytes in mice treated with theEPO-CTP polypeptides are summarized in Table 6 hereinbelow. Theseresults show that EPO-3 is the most potent stimulator of erythropoiesis.

TABLE 6 % reticulocytes Days 2 14 Control 3.72 3.46 1.08 0.8 Mock 3.53.64 0.6 1.13 7010 SEQ ID NO: 16 3.5 3.9 0.6 1.54 7011 SEQ ID NO: 1 3.521.94 1.38 1.08 7012 SEQ ID NO: 2 3.82 3.0 1.02 0.88 7013 SEQ ID NO: 32.66 5.20 0.97 2.96 7014 SEQ ID NO: 4 3.48 3.82 0.71 0.90 Recormon ® 1/W3.23 3.27 0.73 0.59 Recormon ® 3/w 4.13 4.24 1.21 1.14Conclusion

The in vivo experiment was designed to measure two parameters; the firstwas to measure erythropoiesis parameters such as percentage ofreticulocytes and increase of hemoglobin, RBC and hematocrit levels. Thesecond was to measure the durability of the biological activity of eachvariant by injecting once weekly doses.

A superior performance of EPO-3 in its ability to stimulateerythropoiesis was observed in normal mice.

Example 5 Comparison of the EPO-CTP Polypeptides of the PresentInvention to Aranesp®

The following experiment was performed in order to compare thebiological activity of a single bolus dose of some EPO-CTP polypeptidesof the present invention, commercial EPO and Aranesp®. Aranesp® is acommercial long-acting recombinant erythropoietin in which two sitemutations were introduced, resulting in two additional N-glycosylationsites and an increase in the number of incorporated sialic acidresidues.

Materials and Methods

Animals:

-   Species/Strain: Female CD-1 Mice of either sex about 20-25 g-   Group Size: n=3

Experimental design of the study: The experiment was set up assummarized in Table 7 hereinbelow.

TABLE 7 Dose animals/ Solution Dose Group group/ Conc. VolumeTime-Points * # Test Article time-point (μg/mL) (mL/kg) (hourspost-administration) 1 MOD-7010 3 1.5 10 0 (Pre-dose), 0.25, 0.5, 1, 2,6, 24, 48, 96, 168, 216, 264 and 336 hr post-dose administration 2MOD-7013 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, SEQ ID NO: 3 168, 216,264 and 336 hr post- dose administration 3 Aranesp ® 3 1.5 10 0.25, 0.5,1, 2, 6, 24, 48, 96, 168, 216, 264 and 336 hr post- dose administration

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 14 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countand hematocrit examination were performed as described above.

Results

The results are illustrated in FIGS. 6-9. Following a single I.V.injection of 15 μg/kg of EPO 3, all three blood parameters associatedwith erythropoietin i.e. number of reticulocytes, hemoglobin level andhematocrit, were improved relative to those obtained with similarinjected dose of rhEPO or Aranesp®.

Example 6 Comparison of the Pharmacokinetics of EPO-CTP Polypeptides ofthe Present Invention to Aranesp®

The following experiment was performed in order to compare thepharmacokinetics of EPO-CTP polypeptide of the present invention,commercial EPO and Aranesp®.

Materials and Methods

Serum samples were analyzed in order to determine specific concentrationlevels for each sample. Concentration and time-point data were processedusing WinNonLin noncompartmental analysis. Parameters determinedincluded: AUC, CL, Ke, T_(1/2), Cmax, Tmax, and Vdz.

Serum concentrations were determined using two ELISA kits in parallel.EPO-3 serum concentration was measured using StemCell ELISA kit incomparison to EPO-0 and Aranesp® serum concentration which weredetermined using R&D system ELISA kit.

Results

The results of the pharmacokinetic analysis are summarized in Table 8,hereinbelow. These results show that EPO 3 exhibited favorablepharmacokinetic measures as indicated for example in AUC measures, t1/2,and Cmax. Tmax measures were equal to EPO-0, EPO-3, and Aranesp®.

TABLE 8 Parameters Units EPO-0 EPO-3 Aranesp ® AUClast hr*mIU/mL 31739306072 178661 CL{circumflex over ( )} mL/hr/kg 1.1152 0.2188 0.1207 Ke1/hr 0.157 0.0529 0.0639 t½ hr 4.4139 13.1141 10.84 Cmax mIU/mL 1076616466 13266 Tmax Hr 0.25 0.25 0.25 Vdz mL/kg 7.1017 4.1394 1.8877

The results of the serum concentration analysis are illustrated in FIG.9. These results show that EPO-3 was still detectable in the serum afterabout 190 hours. Both EPO-0 and Aranesp® were not detectable in theserum after about 140 hours and 50 hours, respectively.

Conclusion

Clearance of EPO-3 (MOD-7013) from the blood of CD-1 mice wassignificantly slower than that for rhEPO or Aranesp®. The correspondingcalculated half-life times were: rhEPO—4.41 h; Aranesp®—0.84 h; andMOD-7013—13.11 h.

Example 7 Generation of hGH Constructs

Materials and Methods

Four hGH clones (variants of 20 kD protein) were synthesized. XbaI-Not Ifragments containing hGH sequences from the four variants were ligatedinto the eukaryotic expression vector pCI-dhfr previously digested withXbaI-NotI. DNA from the 4 clones (401-0, 1, 2, 3 and 4) was prepared.Another partial hGH clone (1-242 bp) from 22 kD protein was alsosynthesized (0606114). Primers were ordered from Sigma-Genosys. Theprimer sequences used to generate the hGH-CTP polypeptides of thepresent invention are summarized in Table 9 hereinbelow.

TABLE 9 Restric- Pri- tion site mer  SEQ  (under- num- ID lined in  ber NO sequence sequence) 25   27 5′ CTCTAGAGGACATGGCCAC 3′ XbaI 32^(R) 285′ ACAGGGAGGTCTGGGGGTTCTG CA 3′ 33   29 5′ TGCAGAACCCCCAGACCTCCCTGTGC 3′  4^(R) 30 5′ CCAAACTCATCAATGTATCTTA 3′ 25   31 5′CTCTAGAGGACATGGCCAC 3′ XbaI 35^(R) 32 5′ CGAACTCCTGGTAGGTGTCAAA GGC 3′34   33 5′ GCCTTTGACACCTACCAGGAGT TCG 3′ 37^(R) 34 5′ACGCGGCCGCATCCAGACCTTC NotI ATCACTGAGGC 3′ 39^(R) 35 5′GCGGCCGCGGACTCATCAGAAG CCGCAGCTGCCC 3′

Construction of 402-0-p69-1 (hGH) SEQ ID NO: 36: MOD-4020 is the wildtype recombinant human growth hormone (without CTP) which was preparedfor use as control in the below described experiments.

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 32^(R) and plasmid DNA of 0606114 (partialclone of hGH 1-242 bp) as a template; as a result of the PCRamplification, a 245 bp product was formed.

The second reaction was conducted with primer 33 and primer 4^(R) andplasmid DNA of 401-0-p57-2 as a template; as a result of the PCRamplification, a 542 bp product was formed.

The last reaction was conducted with primers 25 and 4^(R) and a mixtureof the products of the previous two reactions as a template; as a resultof the PCR amplification, a 705 bp product was formed and ligated intothe TA cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotIfragment containing hGH-0 sequence was ligated into the eukaryoticexpression vector pCI-dhfr. The vector was transfected into DG-44 CHOcells. Cells were grown in protein-free medium.

Construction of 402-1-p83-5 (hGH-CTP)—SEQ ID NO: 37 and 402-2-p72-3(hGH-CTPx2)—SEQ ID NO: 38: MOD-4021 is a recombinant human growthhormone which was fused to 1 copy of the C-terminal peptide of the betachain of human Chorionic Gonadotropin (CTP). The CTP cassette ofMOD-4021 was attached to the C-terminus (one cassette). MOD-4022 is arecombinant human growth hormone which was fused to 2 copies of theC-terminal peptide of the beta chain of human Chorionic Gonadotropin(CTP). The two CTP cassettes of MOD-4022 were attached to the C-terminus(two cassettes).

Construction of hGH-CTP and hGH-CTP-CTP was performed in the same way asthe construction of hGH-0. pCI-dhfr-401-1-p20-1 (hGH*-ctp) andpCI-dhfr-401-2-p21-2 (hGH*-ctp x2) were used as templates in the secondPCR reaction.

MOD-4021 and MOD-4022 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4021 is ˜30.5Kd since hGH has a MW of 22 Kd while each “CTP cassette” contributes 8.5Kd to the overall molecular weight (see FIG. 10). The molecular weightof MOD-4022 is ˜39 Kd (see FIG. 10).

Construction of 402-3-p81-4 (CTP-hGH-CTP-CTP)—SEQ ID NO: 39 and402-4-p82-9 (CTP*hGH-CTP-CTP)—SEQ ID NO: 40: MOD-4023 is a recombinanthuman growth hormone which was fused to 3 copies of the C-terminalpeptide of the beta chain of human Chorionic Gonadotropin (CTP). Thethree CTP cassettes of MOD-4023 were attached to both N-terminus (onecassette) and the C-terminus (two cassettes). MOD-4024 is a recombinanthuman growth hormone which is fused to 1 truncated and 2 complete copiesof the C-terminal peptide of the beta chain of human ChorionicGonadotropin (CTP). The truncated CTP cassette of MOD-4024 was attachedto the N-terminus and two CTP cassettes were attached to the C-terminus(two cassettes).

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 35^(R) and plasmid DNA of p401-3-p12-5 or401-4-p22-1 as a template; as a result of the PCR amplification, a 265or 220 bp product was formed. The second reaction was conducted withprimer 34 and primer 37^(R) and plasmid DNA of TA-hGH-2-q65-1 as atemplate; as a result of the PCR amplification, a 695 bp product wasformed. The last reaction was conducted with primers 25 and 37^(R) and amixture of the products of the previous two reactions as a template; asa result of the PCR amplification, a 938 or 891 bp product was formedand ligated into TA cloning vector (Invitrogen, catalog K2000-01). XbaI-Not I fragment containing hGH sequence was ligated into our eukaryoticexpression vector pCI-dhfr.

MOD-4023 and MOD-4024 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4023 is ˜47.5Kd (see FIG. 10) and the molecular weight of MOD-4024 is ˜43.25 Kd (seeFIG. 10).

Construction of 402-6-p95a-8 (CTP-hGH-CTP)—SEQ ID NO: 41: Constructionof hGH-6 was performed in the same way as the construction of hGH-3.pCI-dhfr-402-1-p83-5 (hGH-ctp) was used as a template in the second PCRreaction.

Construction of 402-5-p96-4 (CTP-hGH)—SEQ ID NO: 42: PCR reaction wasperformed using primer 25 and primer 39^(R) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp (402-6-p95a-8) as a template; as a result of thePCR amplification, a 763 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). Xba I-Not I fragmentcontaining ctp-hGH sequence was ligated into our eukaryotic expressionvector pCI-dhfr to yield 402-5-p96-4 clone.

Example 8 In Vivo Bioactivity Tests of hGH-CTP Polypeptides of thePresent Invention

The following experiment was performed in order to test the potentiallong acting biological activity of hGH-CTP polypeptides in comparisonwith commercial recombinant human GH and MOD-4020.

Materials and Methods

Female hypophysectomized rats (60-100 g) received a weekly S.C.injection of 21.7 μg hGH-CTP polypeptides or a once daily 5 μg S.C.injection of control commercial rhGH.

Weight was measured in all animals before treatment, 24 hours followingfirst injection and then every other day until the end of the study onday 21. Each point represents the group's average weight gain percentage((Weight day 0-weight last day)/Weight day 0). Average weight gain wasnormalized against once-daily injection of commercial hGH. The treatmentschedule is summarized in Table 10.

TABLE 10 Equimolar Accumulate Dose Treatment Dose Dosage Vol. No. Drug NRoute Schedule (μg/rat) (μg/rat) (ml) 1 Vehicle 7 s.c. days 1, 7 NA NA0.25 and 13; 1/W 2 Mock 7 s.c days 1, 7 NA NA 0.25 and 13; 1/W 3MOD-4020 7 s.c days 1, 7 21.7 65 0.25 SEQ ID NO: 36 and 13; 1/W 4MOD-4021 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 37 and 13; 1/W 5MOD-4022 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 38 and 13; 1/W 6MOD-4023 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 39 and 13; 1/W 7MOD-4024 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 40 and 13; 1/W 8Commercial 7 s.c. days 1, 7 21.7 65 0.25 hGH v.1 and 13; 1/W 9Commercial 7 s.c. days 1-13; 5 65 0.25 hGH v.1 d/WResults

Results are summarized in FIG. 11. These results show that MOD-4023 (SEQID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced over 120% weight gaincompared to commercial rhGH which induced 100% weight gain.

Conclusion

Three weekly doses (Days of injections; 1, 7, and 13) of 21.7 μg ofMOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced a 30%greater weight increase in hypophysectomised rats compared to commercialrhGH injected at the same accumulated dose which was administered onceper day at a dose of 5 μg for 13 days.

Example 9 Pharmacokinetic Studies of CTP-Modified GH

Single-dose pharmacokinetic studies were conducted in Sprague-Dawleyrats. All animal experimentation was conducted in accordance with theAnimal Welfare Act, the Guide for the Care and Use of LaboratoryAnimals, and under the supervision and approval of the InstitutionalAnimal Care and Use Committees of Modigene, Biotechnology General Ltd.Rats were housed either individually or two per cage in rooms with a12-h light/dark cycle. Access to water (municipal supply) andnoncertified rodent chow was provided ad libitum.

To compare the pharmacokinetics of CTP-hGH-CTP-CTP and GH in rats, fourgroups of Sprague-Dawley rats (270-290 g), three to six male rats pergroup. The rats were randomly assigned to four treatment groups (seeTable 11). Rats were administered a single s.c. or i.v. injection of GH(50 μg/kg i.v. or s.c.) or CTP-hGH-CTP-CTP (108 μg/kg i.v. or s.c.).With the exception of the predose sample, which was collected underisoflurane anesthesia, blood collection was performed in unanesthetizedanimals Blood samples (approximately 0.25 ml) were collected inEDTA-coated microtainers for ELISA analyses of CTP-hGH-CTP-CTP plasmaconcentration at the times outlined in Table 11. After each sampling,the blood volume was replaced with an equal volume of sterile 0.9%saline. Samples were stored on ice for up to 1 h prior to centrifugationand plasma harvest. Plasma samples were stored at approximately −20° C.prior to analysis.

TABLE 11 Experimental design of rat pharmacokinetic study No. ofanimals/ Dose Injected Concentration Time-Points * Trt. Test group/ DoseLevel Vol. (μg/ml)/Total (hours post- Grp. Article timepoint RouteGender (μg/kg) (μl) vol. (ml) dose) 1 Biotropin 6# SC Male  50 500 20/5 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48 2 CTP- 6# SC Male 108 500 43.2/5  0.5, 2, 4, 8, hGH- 24, 48, 72, CTP 96 CTP 3 Biotropin 6# IV Male  50 30020/3  0, 0.12, 2, 4, 8, 24 4 CTP- 6# IV Male 108 300 43.2/3   0.12, 2,4, 8, hGH- 24, 48, 72 CTP- CTP Volume of blood sample/time point-500 μlTerminal blood samples # 3 rats per time point.

A commercial sandwich ELISA kit specific for detection of human growthhormone (Roche Diagnostics) was used for evaluation of the rat plasmasamples. This kit detects human growth hormone in plasma by means of anantibody sandwich ELISA format. This kit was initially used to measurethe concentration of CTP-hGH-CTP-CTP in rat plasma. For these plasmasamples, an CTP-hGH-CTP-CTP standard curve (1.2-100 ng/ml) was used andthe concentrations of CTP-hGH-CTP-CTP in rat plasma were interpolatedfrom this curve.

Standard pharmacokinetic parameters, including clearance (CL or CL/F),volume of distribution (Vd or Vd/F), half-life (t_(1/2)), area under theplasma concentration versus time curve (AUC), maximal observed plasmaconcentration (C_(max)) and time to maximal observed plasmaconcentration (T_(max)), were obtained from plasma albutropin or GHconcentration/time curves by noncompartmental analysis using themodeling program WinNonlin (Pharsight, version 3.1). PlasmaCTP-hGH-CTP-CTP or GH concentration data were uniformly weighted forthis analysis. The AUC was calculated using the log-linear trapezoidalanalysis for the i.v. data and the linear-up/log-down trapezoidal methodfor the s.c. data. Plasma concentration profiles for each rat (with theexception of the s.c. albutropin data) or monkey were analyzedindividually, and mean±standard error of the mean (S.E.M.) values forthe pharmacokinetic parameters are reported in Table 12 and FIG. 12.

CTP-hGH-CTP-CTP is a single chain protein of 275 amino acids and up totwelve O-linked carbohydrates. The structure consists of modified humanGrowth Hormone (Somatropin) attached to three copies of the C-terminalpeptide (CTP) of the beta chain of human Chorionic Gonadotropin (hCG);one copy at the N-terminus and two copies (in tandem) at the C terminusHuman Growth Hormone is comprised of 191 amino acids. CTP is comprisedof 28 amino acids and four O-linked sugar chains.

Example 10 Pharmacokinetics of CTP-Modified GH in SD Rats

The pharmacokinetics of CTP-hGH-CTP-CTP was evaluated and compared tothat of commercial hGH (Biotropin).

TABLE 12 Mean pharmacokinetic parameters following single-dose i.v. ands.c. administration of CTP-hGH-CTP-CTP and GH (Biotropin) inSprague-Dawley rats. PK Statistics SC IV CTP- CTP- hGH- hGH- CTP- CTP-Parameters Units Biotropin CTP Biotropin CTP Dose mg/Kg 50 50 50 50AUClast hr*ng/mL 41 680 162.7 1568.3 Cmax ng/ml 13 36.8 275.8 926 Tmaxhr 0.5 8 0 0 MRT hr 2.5 12.9 0.5 9.9 T½ alpha hr 1.58 0.74 T½ beta hr1.73 9 0.5 6.9Data Statistical Analysis

Analysis of serum samples was performed in order to determine specificconcentration levels for each sample. Concentration and time-point datawere processed using WinNonLin noncompartmental analysis.

Parameters that were determined included: AUC, MRT, t1/2, Cmax, andTmax. FIG. 12 demonstrates the superior pharmacokinetic profile ofCTP-hGH-CTP-CTP plasma concentration compared to GH concentrations(pg/ml) following a single i.v. or s.c. dose of CTP-hGH-CTP-CTP or GH inrats (n=3-6 per dose/route).

Following a single S.C. injection of 50 μg/kg, clearance ofCTP-hGH-CTP-CTP from SD rat's blood was significantly slower than thatof CTP-hGH-CTP and of Biotropin. The corresponding calculated half-lifetimes and AUCs were:

Biotropin T½ 1.7 h, AUC 41 hr*ng/mL CTP-hGH-CTP T½ 8.5 h, AUC 424hr*ng/mL CTP-hGH-CTP-CTP T½ 9.0 h, AUC 680 hr*ng/mLConclusion: CTP-hGH-CTP-CTP was chosen as the final candidate out of 6other variants. CTP-hGH-CTP-CTP demonstrated superior performance interms of biological activity and pharmacokinetics.

Example 11 Weight Gain Assay (WGA) for Single Dose/Repeated Dose ofCTP-Modified GH

Hypophysectomized (interaural method) male rats, 3-4 weeks of age, wereobtained from CRL Laboratories. During a post-surgical acclimationperiod of 3 weeks, rats were examined and weighed twice weekly toeliminate animals deemed to have incomplete hypophysectomy evidenced byweight gain similar to that of sham-operated rats. Those rats withincomplete hypophysectomized were eliminated from the study. The averagebody weights of the hypophysectomized were 70-90 grams, at the time ofthe experiment. This is the standard USP and EP bioassay for hGH.Hypophysectomized rats (rats from which the pituitary gland was removed)lose their ability to gain weight. Injections of hGH (and ofCTP-hGH-CTP-CTP) to these rats result in weight gain. Based on themeasured weight gain along a defined period of time and the amount ofhGH injected, the specific activity of hGH (and CTP-hGH-CTP-CTP) isdetermined. Rats were administered either a single s.c. doses of 0.4,0.8 and 4 mg/Kg or repeated s.c. doses of 0.6 and 1.8 mg/Kg 4 days apartfor 3 weeks. Individual body weights of all animals are determined atrandomization, prior to the first dosing, thereafter every two days orin case of decedents at the time of death, and prior to sacrifice.

Single Dose and Repeated Dose Weight Gain Assay

The results comparing whole body growth response following differentdosing patterns of CTP-hGH-CTP-CTP in hypophysectomized rats aredemonstrated in FIG. 13. The results demonstrate that a single injectionof 0.4 & 0.8 mg/Kg/day doses of hGH-CTP were equivalent to 4 dailyinjections of 0.1 mg/Kg/day of Biotropin. The peak of the hGH-CTP effectwas after 2 days.

FIG. 14 further demonstrates that the area under the curve followingsingle injection of CTP-hGH-CTP-CTP correlates with Body Weight gain inRats. Thus, the collective data demonstrates that body weight gain isclosely correlated with cumulative AUC.

The hGH-CTP construct administered 4 days apart promotes the same weightgain as daily injections of Biotropin as demonstrated in FIG. 15.Half-life of hGH in humans is expected to be 5× better than inrats—indicating potential peak effect in humans after 10 days for onesingle injection. These results support administration of hGH-CTPconstruct, CTP-hGH-CTP-CTP, once weekly or biweekly in humans.

Example 12 Pharmacodynamics/Pharmacokinetics Studies of CTP-Modified GH

Hypophysectomized (interaural method) male rats, 3-4 weeks of age, wereobtained from CRL Laboratories. During a post-surgical acclimationperiod of 3 weeks, rats were examined and weighed twice weekly toeliminate animals deemed to have incomplete hypophysectomy evidenced byweight gain similar to that of sham-operated rats. Those rats withincomplete hypophysectomized were eliminated from the study. The averagebody weights of the hypophysectomized and sham rats were 70 and 150 g,respectively, at the time of the experiment.

Rats were administered a single s.c. with CTP-hGH-CTP-CTP, vehicle,human growth hormone CTP-hGH-CTP-CTP or human growth hormone (20 μg/rat)was administered s.c. in an injection volume of 0.2 ml/rat. The dose ofGH was 0.35 and 1.05 μg/Kg, a dose of growth hormone that was equimolarwith the amount of GH in a corresponding 0.6 and 1.8 μg/Kg dose ofCTP-hGH-CTP-CTP. The treatment groups are summarized in Table 13.Following injection, plasma samples for IGF-1 analyses were obtained atthe times described in Table 13. Samples were analyzed for IGF-1concentration using a commercial ELISA (R&D systems).

TABLE 13 Treatment schedule for hypophysectomized rat study CTP- hGH-No. of CTP- animals/ Eq. Eq. CTP Dose Time-Points * Trt. Test group/Dose Dose Dosage Conc. Vol. (hours post- Grp. Article timepoint Route(mg/rat) (mg/Kg) mg/ml (ml) dose) M7 Biotropin  9 SC 0.032 0.35 0.16 0.20 (Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M8 Biotropin  9 SC 0.095 1.050.475 0.2 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M9 EN648-01- 12 SC0.032 0.35 0.275 0.2 1, 2, 4, 8, 24, 08-005 (0.055) (0.6) 48, 72, 96 M10EN648-01- 12 SC 0.095 1.05 0.825 0.2 1, 2, 4, 8, 24, 08-005 (0.165)(1.8) 48, 72, 96 Volume of blood sample/time point-500 μl Terminal bloodsamples

Non-compartmental pharmacokinetic analysis was performed on the meanserum concentration versus time curves for each group. CTP-hGH-CTP-CTPCmax was significantly higher than Biotropin Cmax. The terminalhalf-live of CTP-hGH-CTP-CTP was 6 times higher than Biotropin.

TABLE 14 Pharmacokinetic Parameter Estimates of CTP-hGH-CTP-CTP andBiotropin Following a Single Subcutaneous Injection in hypophysectomizedRats Dose Cmax Tmax AUC_(0-∞) AUC_(0-t) CL/F T_(1/2) Group mg/kg Genderng/mL hr ng-hr/mL ng-hr/mL mL/hr/kg hr CTP- 1.8 M 2,150 8 37,713 37,6950.928 6.86 hGH- CTP- CTP 0.6 M 681 8 11,505 11,489 3.042 6.8 hGH 1.05 M1,078 0.5 3,541 3,540 9.884 1 0.35 M 439 0.5 1,279 1,279 27.36 1

The AUC_(0-t) and the AUC_(0-∞) were very similar suggesting theduration of sampling was adequate to characterize the pharmacokineticprofiles. AUC of CTP-hGH-CTP-CTP was 10 times higher than of Biotropin.Moreover, Cmax was generally proportional to dose and forCTP-hGH-CTP-CTP and it was twice higher than Cmax of Biotropin. However,as shown in FIG. 16, Tmax of CTP-hGH-CTP-CTP was 8 hr as compare to 0.5hr of Biotropin, and the terminal half-lives did not appear to vary withdose level. T1/2 of CTP-hGH-CTP-CTP was 6.8 times longer than ofBiotropin.

Indirect effects of GH are mediated primarily by an insulin-like growthfactor-I (IGF-I), a hormone that is secreted from the liver and othertissues in response to growth hormone. A majority of the growthpromoting effects of growth hormone is actually due to IGF-I acting onits target cells. Accordingly, the effect of the CTP-hGH construct,CTP-hGH-CTP-CTP, on IGF-1 serum levels in Hypophysectimized Rats wasmeasured. FIG. 17 presents results of IGF-1 serum levels inHypophysectimized Rats Following SC injection of CTP-hGH-CTP-CTP andcommercial hGH.

Single dose of CTP-hGH-CTP-CTP 0.6 or 1.8 mg/Kg and Biotropin 0.35 or1.05 mg/Kg were injected subcutaneously to hypophysectomised rats fordetermination of PK/PD profile. Serum IGF-I post injection was measuredusing specific ELISA kits (Roche Diagnostics).

The cumulative serum levels of IGF-I following injection ofCTP-hGH-CTP-CTP was significantly higher than following injection ofBiotropin. Cmax was generally proportional to dose and forCTP-hGH-CTP-CTP it was 3-4 times higher than Cmax of Biotropin. Tmax ofCTP-hGH-CTP-CTP was 36-48 hr as compare to 20-24 hr of Biotropin. Inconclusion, higher hGH levels and longer presence in serum result insignificant increase in IGF-1 levels.

Example 13 Carbohydrate Content and Sialic Acid Content of CTP-ModifiedGH

Analysis of O-glycans is based on a Prozyme kit. O-glycans arechemically and enzymatically cleaved from the protein and separated frompeptides using paper chromatography. Sequencing of the O-glycan pool isperformed by sequential enzymatic digestions (exo-glycosidases) followedby HPLC analysis compared to standards.

Glycoprofiling with Sequence Analysis

Glycoprofiling was performed by Ludger Ltd. Two samples (EN648 andRS0708) were taken through triplicate releases and each release was alsoanalyzed by HPLC in triplicate. Triplicate 300 μg samples of EN648 andRS0708 and a single 100 μl sample of citrate/sodium chloride buffer,plus a positive control fetuin (250 μg) and a 100 μl water negativecontrol, were ultra-filtrated by centrifugation using a molecular weightcut off membrane of 10,000 Da to replace the buffer with water, thentaken through hydrazinolysis under O-mode conditions (6 h at 60° C.).Released glycans were re-N-acetylated and cleaned up by LudgerClean CEXcartridges. An aliquot of the released glycans was then labeled with2-aminobenzamide (2AB), cleaned up with Ludger Clean S cartridges andanalyzed by LudgerSep-N2 HILIC-HPLC.

Monosaccharide Content

Analysis of neutral monosaccharides requires hydrolysis of glycans totheir constituent monosaccharide components. The hydrolysis wasperformed by Ludger Ltd, on intact glycoprotein samples. Specifically,50 μg of intact glycoprotein was acid hydrolyzed, 2-AB(2-aminobenzamide) labeled and run on a reverse phase HPLC column. Thismethod hydrolyzes all glycans present on the glycoprotein inclusive of Nand O linked types.

Sialic Acid Profiling

Two samples (EN648 and RS0708) and a buffer control were analyzed.Sialic acid analysis requires mild acid release of the monosaccharidesfollowed by DMB fluorophore labeling and HPLC analysis on a LudgerSep-R1column 50 μg of intact glycoprotein was acid hydrolyzed for eachanalysis.

Glyco Analysis of CTP-hGH-CTP-CTP

TABLE 15 Glycan analysis. Structural assignments and percentage areas ofpeaks are based upon HPLC and enzyme array digests. Percent from totalglycans^(e) Peak ABS ID^(a) GU^(b) Structure^(c) name Und^(d) NAN1 ABSBTG 1^(f) 0.92

GalNAc 0.4 0.4 0.6 53.0 2^(f) 1.02

galactose 1.9 9.7 23.8 26.5 * 1.72 4.3 4.6 2.3 3 1.79

Galβ1-3GalNAc 2.3 67.7 69.4 17.1^(h) 4^(g) 2.25

NeuNAcα2-3Gal 19.8 13.0^(h) * 2.57 1.5 1.9 1.1 1.1 5 2.90

NeuNAcα2-3Galβ1-3 GalNAc 70.6 * 3.58 0.6 0.7 0.6 6 3.22

Galβ1-3[NeuNAcα2-6] GalNAc 0.9 2.3 7 4.42

NeuNAcα2-3Galβ1-3 [NeuNAcα2-6]GalNAc 1.8

The monosaccharide profiles indicate that the CTP-hGH-CTP-CTPglycoprotein samples contain predominantly O-link type glycans. Themajor O-glycan peak is sialylated core 1 (Neu5Acα2-3Galβ1-3GalNAc). Themajor sialic acid is Neu5Ac and there are some minor peaks suggestingthe presence of 3-4% of di-acetylated sialic acidN-acetyl-9-O-acetylneuraminic acid (Neu5, 9Ac2) and less than 1%N-glycolylneuraminic acid. There are also small amounts ofNeu5Acα2-6(Galβ1-3)GalNAc.

Example 14 Pharmacokinetic/Toxicokinetic Analysis of CTP-Modified GH inRhesus Monkeys

Serum concentrations versus time curves were generated for each animal.Non-compartmental analysis was performed with WinNonlin professionalversion 5.2.1 (Pharsight Corporation, Mt View Calif.). The estimatedpharmacokinetic parameters are shown in Table 16 below:

TABLE 16 Estimates of CTP-hGH-CTP-CTP Pharmacokinetic Parameters (Mean ±SD) from Non-compartmental Analysis Following A Single SubcutaneousInjection in Rhesus Monkeys Parameter 1.8 mg/kg 90 mg/kg Cmax (μg/mL) 2.073 ± 0.417 108.7 ± 46.0 Tmax (hr)  4 ± 0 11 ±7  AUC_(0-t) (μg-hr/mL)38.7 ± 7.4 2,444 ± 394  AUC_(0-∞) (μg-hr/mL) 39.0 ± 7.3 2,472 ± 388 CL/F (mL/hr/kg) 47.5 ± 9.0 37.04 ± 4.78 T_(1/2) (hr) 10.00 ± 1.47  9.85± 1.07 Vz/F (mL/kg)  701 ± 236  529 ± 104

The AUC_(0-t) and the AUC_(0-∞) were very similar suggesting theduration of sampling was adequate to characterize the pharmacokineticprofiles. Cmax was proportional to dose. Tmax was later at the higherdose. Tmax was at 4 hours for all animals in the low dose group and wasat 8 or 24 hours in the high dose group. Terminal half-lives are similarfor the two dose groups.

AUC was approximately proportional to dose with a slightly larger thanproportional AUC at the higher dose producing a slightly lower estimatefor CL/F and Vz/F compared to the lower dose. It is not possible to sayif CL and Vz are lower at the higher dose or if F is lower at the lowerdose. There was overlap between the groups so it is questionable thatthis represents a meaningful difference in CL/F and Vz/F.

Pharmacokinetic parameters estimated by the model were very similar tothose from non-compartment analysis. Absorption and eliminationhalf-lives are shown in Table 17 below:

TABLE 17 Estimates of CTP-hGH-CTP-CTP Absorption and EliminationHalf-lives (Mean ± SD) Following Subcutaneous Injection Derived FromPharmacokinetic Modeling in Rhesus Monkeys Dose T_(1/2 abs) (hr)T_(1/2 el) (hr) 1.8 mg/kg 1.17 ± 0.40 10.41 ± 2.36 90 mg/kg 6.49 ± 1.87 7.26 ± 1.85

The data indicate that the elimination rates are fairly similar betweenthe groups with a slightly longer T1/2 el in the lower dose group. Theabsorption, however, is more than 5-fold slower following subcutaneousadministration of 90 mg/kg compared to that following 1.8 mg/kg. As inthe case of the non-compartmental analysis, modeling indicated a laterTmax at the high dose.

Although GH supplementation is effective in the treatment of GHdeficiency in children and adults, the disadvantages of daily injectionsover extended periods of time limit its use by physicians in certainpatient populations as well as increase the risk of dosing error, thenumber of care givers, the cost of treatment and/noncompliance.Especially important in certain populations, such as children of shortstature who may not understand the implications of not following theprescribed GH dosing regimen, is the necessity of compliance to achievethe optimal benefit from GH therapy. The issue of finding a moresuitable alternative to daily GH injections and subsequent compliancegains further importance as GH-deficient children transition into adultswith a continuing need for GH treatment. The requirement of dailytherapy is largely due to recombinant GH's short plasma half-life andhas led to the development of a sustained release form of GH (Reiter EO. Attire K M., Mashing T J. Silverman B L. Kemp S F. Neolith R B. FordK M. and Sanger P. A multimember study of the efficacy and safety ofsustained release GH in the treatment of naive pediatric patients withGH deficiency. J. Clin. Endocrinol. Metab. 86 (2001), pp. 4700-4706).

GH-CTP, a recombinant human growth hormone-CTP fusion protein, asdescribed herein, has a pharmacokinetic profile in the rat that islonger in duration than that of GH. This unique pharmacokinetic profileallows for intermittent administration of GH-CTP to achievepharmacodynamic effects in growth-hormone-deficient rat as evidenced bygrowth and elevations in plasma IGF-1 levels, respectively.

GH-CTP offers a superior pharmacokinetic profile compared with that ofGH when administered s.c. in the rat. There are substantial differencesin plasma clearance of GH-CTP compared to GH. Specifically, plasma iscleared of GH-CTP at more than 6 times more slowly than GH followings.c. dosing. The terminal half-life and mean residence time of GH-CTPwere approximately six times longer than that of GH in rats followings.c. administration. In addition, the Cl/F following s.c. dosing is 10times lower for GH-CTP than for GH.

In an effort to examine whether the pharmacokinetic advantages in therat translated to a pharmacodynamic benefit, the possibility that GH-CTPmight stimulate growth in GH-deficient hypophysectomized rats withdosing regimens less frequent than daily was tested at equimolarCTP-hGH-CTP-CTP and GH dose levels. Single SC injection of GH-CTPpromoted incremental weight gain which was equal to 4 daily consecutiveinjections of GH. In addition, the every fourth day administrationschedule for GH-CTP shows enhanced body weight gain over GH.

Pharmacodynamically, the long circulation time of GH-CTP relative to GHin the hypophysectomized rats resulted in a prolonged IGF-1 responsemeasured in blood plasma following a single s.c. injection. Subcutaneousadministration of a single dose of GH-CTP increased circulating IGF-1concentrations in a dose-dependent manner in the hypophysectomized rats.At the highest albutropin dose, IGF-1 concentrations were elevated abovebaseline for as long as 75 hours after a single administration. Thus,the enhanced circulation time of a single dose of GH-CTP resulted insubstantial pharmacodynamic improvement over a single dose of GH,raising the possibility that GH-CTP could offer similar growthenhancement with reduced dosing frequency compared with standard GHtreatment regimens.

Single CTPs modified hGH-dose of 90 mg/kg in Rhesus and 180 mg/kg inrats were well tolerated in both species. The allometric factor betweenrats and primates is approximately ×2 which is based on the anticipatedclearance of proteins in these animals. In-line with industry-acceptedextrapolation models for therapeutic proteins' half-life increasebetween species (FDA Guidance). 90 mg/kg in Primates has a PK profileslightly better than 180 mg/kg of CTPs modified hGH in Rat. Thus,allometric extrapolation to humans supports weekly or once/2 winjection.

The present concept utilizing a CTP-GH construct, reduced dosingfrequency compared to the commercial GH recombinant product. NutropinDepot® is a sustained release formulation of GH approved for use inpediatric populations; however, comparisons to historical controls haverevealed that 1- and 2-year growth rates are significantly (p<0.001)lower in children given Nutropin Depot® (1-year growth rate 8.2±1.8cm/year) than in children treated with GH (one-year growth rate 10.1±2.8cm/year) (Silverman B L. Blethen S L. Reiter E O. Attie K M. Neuwirth RB. and Ford K M. A long-acting human growth hormone (Nutropin Depot®):efficacy and safety following two years of treatment in children withgrowth hormone deficiency. J. Pediatr. Endocrinol. Metab. 15 (2002), pp.715-722). The local effects of subcutaneously administered NutropinDepot® include nodules, erythema, pain at the injection site, headacheand vomiting. Preclinical toxicology studies in both rat and monkey haveshown that s.c. administration of CTP-hGH-CTP-CTP produces no localreactions compared to vehicle. Given the medical need for a lessfrequently administered form of GH, the pharmacologic properties ofCTP-hGH-CTP-CTP in this study in rats suggest that this product isfavorable also in terms of toxicology and patient compliance. Thesustained activity of CTP-hGH-CTP-CTP in the rat support its potentialutility as an agent that requires only intermittent administration toattain a therapeutic benefit that is currently achieved with dailydosing.

Example 15 Long-Acting CTP-Modified Version of Human Growth Hormone(hGH-CTP) was Highly Effective in Growth Hormone Deficient Adults—PhaseII Clinical Trial

A randomized, open-label, Phase II Clinical Trial was conducted toevaluate the safety, tolerability, pharmacokinetics and pharmacodynamicproperties of hGH-CTP injected either weekly or twice-monthly inpatients who currently receive daily injections of growth hormone. Thetrial was conducted at multiple sites in six countries. The three maincohorts in the trial received a single weekly dose of hGH-CTP,containing 30%, 45% or 100% of the equivalent cumulative commercial hGHdose that growth hormone-deficient adult patients receive over thecourse of seven days in the form of daily injections (referred to as the“30%”, “45%” and “100%” cohorts, respectively). The data reflect resultsfrom 39 patients, 13 in each cohort. 2 females were included in eachcohort.

In addition to the three main cohorts, growth hormone deficient adultswere enrolled in an experimental fourth cohort, which is conductedoutside of the formal Phase II trial. The patients in the experimentalfourth cohort receive a single injection of hGH-CTP once every two weeksthat contains 50% of the cumulative commercial dose of that growthhormone-deficient adult patients receive over a two-week period in theform of daily injections.

Efficacy for the three main cohorts receiving a single weekly injectionof hGH-CTP is defined by measuring daily insulin-like growth factor 1(IGF-1) levels within the desired therapeutic range over a period ofseven days (during the last week of treatment in the study). The desiredtherapeutic range is defined as between +2 standard deviations through−2 standard deviations from the average IGF-1 levels expected in anormal population, stratified by age group and gender. In addition, thetrial measured IGF-1 levels within a narrower range of +/−1.5 standarddeviations for the purpose of observing the variance of the patientswithin the normal range.

Results:

Table 18 contains the average percent of days within the normaltherapeutic range (+/−2 SD), average percent of days within a narrowernormal therapeutic range (+/−1.5 SD), and average Cmax (highestconcentration level) of IGF-1 for males, measured during the lasttreatment week, expressed in standard deviations from the normalpopulation mean IGF-1 levels.

TABLE 18 Human Phase II Clinical Trial Results. % Days Within % DaysWithin Avg. Cmax Narrow Normal Normal Range of IGF-1 Range of IGF-1 ofIGF-1 (preferred Cohort (+/−1.5 SD) (+/−2 SD) below +2 SD) 30% 57% 100%−0.9 45% 100%  100% 0.1 100%  86% 100% 0.4

Two mg per week of hGH-CTP, containing 50% of the cumulative weekly hGHdose that an adult patient would usually be prescribed as the initialtreatment dose, has a high likelihood of being defined as the startingdose for males and females in the adult Phase III.

There was no evidence of safety and/or tolerability issues, and noindication that hGH-CTP, when used in high doses, induced excessivelevels of IGF-1 in patients or even levels above the normal range.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to the precise embodiments, and that various changes andmodifications may be effected therein by those skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

What is claimed is:
 1. A method of treating a subject suffering from afatigue syndrome following cancer chemotherapy or a chronic infection,wherein said subject is anemic, said method comprising administering toa subject a therapeutically effective amount of a polypeptide consistingof a chorionic gonadotropin carboxy terminal peptide (CTP) modifiederythropoietin (EPO), said CTP-modified EPO consisting of one chorionicgonadotropin carboxy terminal peptide (CTP) attached to the aminoterminus of said EPO, and two chorionic gonadotropin CTPs attached tothe carboxy terminus of said EPO, wherein said polypeptide consists of aprecursor or a mature polypeptide and optionally a signal peptideattached to the amino terminus of said one CTP, wherein saidadministration treats the anemic subject suffering from fatigue syndromefollowing cancer chemotherapy or a chronic infection, or any combinationthereof.
 2. The method of claim 1, wherein the sequence of at least onechorionic gonadotropin carboxy terminal peptide consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 17 andSEQ ID NO:
 18. 3. The method of claim 1, wherein at least one chorionicgonadotropin carboxy terminal peptide is truncated.
 4. The method ofclaim 1, wherein said EPO is glycosylated.
 5. The method of claim 1,wherein said EPO is non-glycosylated.
 6. The method of claim 1, whereinat least one chorionic gonadotropin carbon terminal peptide isglycosylated.
 7. The method of claim 1, wherein the amino acid sequenceof said mature CTP-modified EPO polypeptide is set forth in SEQ ID NO:51.
 8. The method of claim 1, wherein the amino acid sequence of saidprecursor CTP-modified EPO polypeptide is set forth in SEQ ID NO:
 3. 9.The method of claim 8, wherein said precursor CTP-modified EPOpolypeptide comprises a signal peptide attached to the amino terminus ofsaid one CTP, wherein the amino acid sequence of said signal peptide isset forth in SEQ ID NO:
 19. 10. The method of claim 1, wherein saidchronic infection comprises HIV, inflammatory bowel disease, or septicepisodes, or any combination thereof.